1 5-Hydroxytryptamine (5-HT) (5.16-1291 microM) produced a phasic contraction followed later by rhythmic contractions in the rat vas deferens, primarily in the epididymal half. 5-HT (129 microM) produced no response in Ca2+-free solution. Nifedipine (0.29 microM) or verapamil (2.04 microM) inhibited the initial phasic response to 5-HT, but inhibition of the rhythmic contractions required concentrations 5 fold (nifedipine) or 30 fold (verapamil) higher. 2 Methysergide (2.13 microM) abolished the phasic and reduced the frequency of the rhythmic contractions. Phentolamine (2.65 microM) did not affect the phasic response but reduced the amplitude of the rhythmic contractions. The combination of phentolamine (2.65 microM) and methysergide (2.13 microM) completely abolished the response to 5-HT (129 microM). 3 Desipramine (1.32 microM) had no effect on the phasic response to 5-HT (129 microM), but the rhythmic contractions were reduced in amplitude with no effect on their frequency. 4 In vasa deferentia removed from reserpine-treated or from guanethidine-denervated rats, both phasic and rhythmic components of the 5-HT (129 microM) contraction were augmented due to supersensitivity. 5 It is concluded that the phasic component of the 5-HT contraction is mediated by post-junctional 5-HT receptors, while the rhythmic component is mediated by the combination of post-junctional 5-HT receptors and noradrenaline released from neuronal stores. Assuming that nifedipine and verapamil are acting solely by inhibition of calcium channels, the phasic and rhythmic components of the 5-HT response may be mediated through separate Ca2+ channels. If this is correct, one channel might be a voltage-dependent channel and the other could be similar to, but distinct from the channel mediating the response to methoxamine.