We have recently shown that the postjunctional α2-adrenoceptor mediating contraction of porcine pulmonary veins is of the α2C-subtype. We could also demonstrate that α1-adrenoceptors might contribute to the contraction in that blood vessel. In the present study, we aimed at characterising the α1-adrenoceptor subtype(s) involved using pharmacological and molecular biological methods. In isolated rings of porcine pulmonary veins the typical α1-adrenoceptor agonist phenylephrine caused a concentration-dependent contraction that was inhibited by the α1B-adrenoceptor selective antagonists 1-[4-(4-amino-6,7-dimethoxyquinazolin-2-yl)piperazin-1-yl]-2-[2-(isopropyl)-6-methoxyphenoxy]ethan-1-one (Rec15/2615; pA2 8.96 ± 0.13) and 4-amino-2-[4-[1-(benzyloxycarbonyl)-2(S)-[[(1,1-dimethylethyl)amino]carbonyl]-piperazinyl]-6,7-dimethoxyquinazoline (L-765,314; pA2 7.22 ± 0.05), as well as the α1D-adrenoceptor selective antagonist 8-[2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl]-8-azaspiro[4.5]decane-7,9-dione (BMY7378; pA2 8.29 ± 0.15, slope of the Schild plot 0.75 ± 0.09, significantly different from unity, P < 0.05), but not by the α1A-adrenoceptor selective antagonists (±)-1,3,5-trimethyl-6-[[3-[4-((2,3-dihydro-2-hydroxymethyl)-1,4-benzodioxin-5-yl)-1-piperazinyl]propyl]amino]-2,4(1H,3H)-pyrimidinedione (B8805–033) and N-[2-(2-cyclopropylmethoxyphenoxy)ethyl]-5-chloro-α,α-dimethyl-1H-indole-3-ethanamine (RS-17053). These findings suggest that phenylephrine activates both α1B- and α1D-adrenoceptors. The observation was confirmed by reverse-transcriptase polymerase chain reaction (RT-PCR) in porcine pulmonary veins, where mRNA signals for α1B- and α1D-adrenoceptors could be detected. However, the antagonist properties of rauwolscine and yohimbine (non-subtype selective α2-adrenoceptor antagonists) against phenylephrine showed that this agonist also activates α2-adrenoceptors in pulmonary veins. This was strengthened in experiments using tissues that were stimulated with forskolin (cell permeable activator of adenylyl cyclase). Phenylephrine mimicked the effect of the selective α2-adrenoceptor agonist UK14304 by causing an inhibition of forskolin-stimulated cAMP accumulation that was blocked by rauwolscine. It is concluded that, in addition to α1B- and α1D-adrenoceptors, phenylephrine can stimulate α2-adrenoceptors in porcine pulmonary veins.