The aims of this investigation were (1) to functionally characterize the α1-adrenoceptor mediating contractions of the rat isolated epididymal vas deferens, spleen and human prostate using α1-adrenoceptor subtype selective antagonists, and (2) to study the possible mechanisms of contraction to α1-adrenoceptor stimulation in these tissues. Contractions mediated by α1-adrenoceptors in the rat epididymal vas deferens and human prostate were antagonized by subtype selective competitive antagonists with pA2 values consistent with their affinities in binding studies on tissue α1A-adrenoceptors and correlated best with the antagonists published affinities for the expressed α1a-adrenoceptor clone. For the rat spleen α1-adrenoceptor mediated contractions the antagonist pA2 values were consistent with their affinities in binding studies on tissue α1B-adrenoceptors and correlated best with the antagonists published affinities for the expressed α1-adrenoceptor clone. It was concluded that the functional α1-adrenoceptor in the rat epididymal vas deferens and human prostate was the α1A-subtype and this is the same as the expressed α1a-clone, while in the rat spleen it was the α1B-subtype and this is the same as the expressed α1b-clone. The α1A-adrenoceptor mediated contraction of the rat epididymal vas deferens appeared to depend upon activation of protein kinase C by diacylglycerol, resulting in the influx of extracellular Ca2+ through voltage-gated Ca2+ channels. The α1B-adrenoceptor mediated contraction of the rat spleen consisted of an initial phasic contraction due to release of intracellular Ca2+ and a larger tonic contraction due to capacitative Ca2+ influx through non-voltage-gated Ca2+ channels and which may involve a tyrosine kinase. The α1A-adrenoceptor mediated contraction in the human prostate was dependent on both influx of extracellular Ca2+ through a partially nifedipine sensitive channel and a component which may possibly be due to either release of Ca2+ from ryanodine sensitive intracellular stores or a Ca2+ independent mechanism.