The men from Millbrae made the long trip to take on their opponents in Pool Six and were well in the match against the men from the English Championship.
At half time the Scottish side, making a return to the cross-border event having missed out last season, were 10-7 up, but red cards to prop George Hunter and back-row Andy Dunlop did not help their cause as they eventually went down 28-16.
Ayr's points in the loss all came from stand-off Finn Russell who bagged a try, three penalties and a conversion.
Captain Tom Brown scored a try for the hosts as did stand-off Aaron Penberthy, prop David Fidler and winger James Copsey. Penberthy and replacement Niall O'Connor shared the kicking points.
Edinburgh Accies battled gamely in their debut match in the competition at home to Welsh Premiership side Pontypridd, but easily lost out 53-13.
The visitors, cheered on by a noisy travelling support, scored two tries in the first four minutes to take control of this one.
Winger Matthew Nuthall got the first and was soon followed by one from stand-off Simon Humberstone to make it 12-0.
Accies then got themselves into things with a penalty by stand-off Alex Blair and a try by prop Phil Cringle before the Welshmen added two more tries before the break.
That meant they went into the interval at Raeburn Place 27-8 to the good. After the break the visitors scored another four tries, including one from centre Gavin Dacey on his 200th appearance for them, as they got over the 50 point mark. Accies' only try after the break came from Blair, but the end score did not reflect how well they did in patches.
In the same pool London Scottish, with former Scotland under-20 captain Lewis Calder playing hooker for the first time, defeated London Welsh.
In-form winger Miles Mantella ran in two tries to lead the way as Scottish came out on top 32-26.
Stirling County played Munster A in Ireland, but were on the wrong end of a 39-8 defeat.
In the Pool Four encounter, County's young squad kept going right until the final whistle, but the home side ran in six tries to come out on top.
In Pool Five, Gala were defeated at home 31-17 by Welsh side Aberavon.
In the RBS Premiership, leaders Melrose were on top form to defeat Aberdeen Grammar 42-16 at the Greenyards.
Scotland under-20 winger Damien Hoyland continued his good season with two tries while five other players helped themselves to tries for the Borderers.
They were props Gary Holborn and Nick Beavon, back-row Peter Eccles, centre Joe Helps and winger Callum Anderson while Helps and fellow centre Andrew Skeen kicked the rest of the points. Prop Struan Cessford got Grammar's only try on a bad day at the office.
Heriot's are still in second spot after edging out Glasgow Hawks 18-13 at Old Anniesland.
Centres Keith Buchan and Max Learmonth scored both Heriot's tries while scrum-half Graham Wilson kicked eight points.
Winger Haddon McPherson and second-row Andy Linton grabbed the tries for the home side with stand-off Scott Wight kicking one penalty as they picked up a losing bonus point.
Currie were cut further adrift at the bottom of the table after they were beaten by Hawick 21-12 at Mansfield Park.
Stand-off Lee Armstrong, back-row Bruce McNeil and winger Greg Anderson scored tries for the Greens that were all converted by full-back Neil Renwick.
Currie, who are stuck on six points in 10th spot, scored tries through teenage full-back Ruairidh Smith and prop John Cox with stand-off George Horne converting one, but it was not enough.
For Hawick it was important to get back to winning ways after a heavy defeat to Ayr last week.
Second-row Greg Scott scored three tries for Boroughmuir as they defeated bottom side Biggar 50-3 and stayed out on top in the RBS National League.
Second-placed Watsonians got back to winning ways by defeating GHA 19-8 at Myreside.