Abstract The effects of clonidine on the purinergic inhibition of cholinergic transmission were investigated; we used the twitch contraction of a circular segment of the guinea-pig Oddi sphincter stimulated electrically. A circular segment of Oddi sphincter was contracted in a frequency-dependent manner by transmural nerve stimulation. Adenosine (10 −6 to 10 −4) produced a reduction of approximately 40% in the twitch contraction elicited at 5 Hz. The inhibition was antagonized by theophylline (10 −5 M), 8-phenyltheophylline (10 −6 M) or clonidine (10 −6 to 3 × 10 −6 M) but was enhanced by dipyridamole (3 × 10 −6 M). This antagonism produced by clonidine (3 × 10 −6 M) was almost unaffected by yohimbine (3 × 10 −6 M). The twitch contraction was also suppressed by N 6-substituted adenosine analogs, L-PIA and CHA. The suppression was counteracted by theophylline or clonidine. The contraction produced by exogenously administered acetylcholine was not affected by clonidine and/or adenosine at all. The results suggest that clonidine, like theophylline, seems to exert these antagonistic effects by acting on the neuronal adenosine receptors in the guinea-pig Oddi sphincter muscle.