In the isolated rat vas deferens, various 2-substituted adenosine derivatives and adenosine inhibited contractions elicited by field stimulation but had little effect on responses to exogenous noradrenaline. 2-Chloroadenosine, 2-bromoadenosine, 2-hydroxyadenosine and 2-fluoroadenosine were all more potent than adenosine. Theophylline antagonized the action of the 2-substituted derivatives. The inhibitory action of adenosine was potentiated by dipyridamole, 2-amino-6-[(2-hydroxy-5-nitro)benzylthio]-9-beta-D-ribofuranosylpurine (HNBTG) or 2'-deoxycoformycin while that of 2-choloroadenosine was not altered by any of these drugs or by phenoxybenzamine, atropine or indomethacin. Pretreatment of the vas deferens with both HNBTG and 2'-deoxycoformycin eliminated the difference in inhibitory potency between adenosine and 2-chloroadenosine. These results indicate that 2-substituted adenosine derivatives, like adenosine, produce inhibition of transmission by acting on a presynaptic site which can be blocked by theophylline. The high apparent potency of 2-chloroadenosine compared to adenosine appears to be due to the former being neither taken up nor deaminated, while the apparent potency of adenosine is masked by uptake and deamination in this tissue.