The ability of a series of N6-modified N-alkyl-5′-uronamides to cause presynaptic inhibition of transmitter release was examined in isolated guinea-pig ileum stimulated at 0.2 Hz. These analogs inhibited the twitch responses to nerve stimulation, the majority being full agonists with their inhibitory effects being antagonised by theophylline. These analogs had no significant effects on responses of ileum to carbachol. N-ethyl 5′-uronamide substitution resulted in an up to four-fold reduction in activity of N6-substituted adenosine analogs, while stereoselectivity of the N6-substituted analogs continued to be present. 5′-Uronamide substitutions to N6-(3-pentyl)-adenosine resulted in a marked loss of activity when there were large alkyl groups at the amide or with amides of secondary amines. It was concluded that adenosine analogs interact with both the N6 and C-5′ regions of the adenosine receptor in this tissue, with the interaction being less than additive.