This study was designed to characterise the muscarinic receptor subtype responsible for acetylcholine-mediated in vitro pulmonary artery relaxation in rats and the importance of the presence of neostigmine (an anti-cholinesterase) during receptor characterisation. Cumulative administration of acetylcholine elicited concentration-dependent relaxation of phenylephrine (1 microM) precontracted preparations. Inclusion of neostigmine (10 microM) caused a parallel leftward shift with an increase of the pD(2) value (7.09 vs. 6.43) of the concentration-response curve of acetylcholine. The magnitude of maximum relaxation, however, was not affected. Using a range of conventional muscarinic receptor antagonists (atropine, pirenzepine, methoctramine, p-FHHSiD and tropicamide) and the highly selective Green Mamba muscarinic toxins (MT-3 and MT-7), it was found that muscarinic M(3) receptors are probably responsible for endothelium-dependent relaxation of the pulmonary artery upon acetylcholine challenge. Preincubation with N(G)-nitro-L-arginine methyl ester (L-NAME, 20 microM, a nitric oxide synthase inhibitor), but not N(G)-nitro-D-arginine methyl ester (D-NAME, 20 microM), abolished acetylcholine-elicited relaxation. Moreover, 6-anilino-5,8-quinolinedione (LY 83583, 1 microM) and methylene blue (1 microM) (both are guanylate cyclase inhibitors) markedly attenuated acetylcholine-elicited relaxation. However, the presence of indomethacin (3 microM, a cyclo-oxygenase inhibitor), (-)-perillic acid (30 microM, a p21(ras) blocker), 2-[2'-amino-3'-methoxy-phenyl]-oxana-phthalen-4-one (PD 98059) (10 microM, a p42/p44 mitogen-activated protein kinase inhibitor), 4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)1H-imidazole (SB 203580) (1 microM, a p38 mitogen-activated protein kinase blocker), wortmannin (500 nM, a phosphatidylinositol-3 kinase inhibitor) and genistein (10 microM, a tyrosine kinase blocker) failed to alter acetylcholine-provoked pulmonary arterial relaxation. These results suggest that acetylcholine caused pulmonary arterial relaxation through the activation of muscarinic M(3) receptors in the endothelium. Moreover, the p21(ras)/mitogen-activated protein kinase pathway seems to play no role in mediating acetylcholine-elicited relaxation.