1 Electrically (30 Hz) induced contractions of guinea-pig isolated ileal longitudinal muscles were reduced by tetrodotoxin (1 micron), adenosine (30 micron) and morphine (10 micron). 2 When stimulated with 10 or 30 Hz for 10 s at 1 min intervals, a progressive decline of amplitude of the contraction was seen (development of tachyphylaxis). At this time, the contractile response to 1,1-dimethyl-4-phenylpiperazinium iodide (DMPP) (10 micron) was also greatly reduced. 3 The smaller responses to electrical stimulation and DMPP during tachyphylaxis were restored to their initial amplitude by the addition of theophylline (10 micron). The appearance of tachyphylaxis was prevented by pretreatment with theophylline (1 to 10 micron) and was greatly accelerated by pretreatment with dipyridamole (0.1 1 micron). 4 In [14C]-choline or [3H]-adenosine preloaded muscle strips, electrical stimulation (30 Hz) increased the 14C- or 3H-output, the effect being sensitive to tetrodotoxin blockade. The tachyphylaxis to electrical stimulation was accompanied by a considerable and sustained increase in 3H-output, an effect that was accelerated by dipyridamole (1 micron). The 14C-output initially increased but fell off gradually with the development of tachyphylaxis at which time theophylline (30 micron) reversed the fall. 5 There was a marked increase in the proportion of released [3H]-adenosine to its derivatives during the development of tachyphylaxis. Approximately 60% of the released total radioactivity after tachyphylaxis was found to be [3H]-adenosine. 6 These results suggest that the development of tachyphylaxis may be closely associated with the release of endogenous adenosine derivatives (mostly adenosine) which have presynaptic inhibitory actions on the cholinergic elements in guinea-pig ileum.