We have examined the pharmacological subtypes of muscarinic receptors mediating phosphoinositide hydrolysis and contraction in the longitudinal smooth muscle of guinea pig ileum with the use of muscarinic antagonists. Carbachol increased the formation of 3H-inositol phosphates (IPs) in a dose-dependent manner in both ileal smooth muscle and the frontal cortex of rats. The rank order of muscarinic antagonists for inhibition of IP formation induced by carbachol was 4-DAMP = atropine > pirenzepine > AF-DX 116 in guinea pig ileal smooth muscle. In ileal smooth muscle, the inhibition by the M1 antagonist pirenzepine was about 15 times less than that by atropine. However, in the rat frontal cortex, the inhibition by pirenzepine was only about 3 times less than that by atropine. The inhibitory effect of the M2 antagonist AF-DX 116 was weak in both ileal muscle and the frontal cortex. The M3 antagonist 4-DAMP strongly inhibited carbachol-induced IP formation in ileal smooth muscle. The rank order of muscarinic antagonists for inhibition of contraction induced by 10(-7) M carbachol was atropine > or = 4-DAMP > pirenzepine > AF-DX 116. These results suggest that both IP formation and the contractile response induced by muscarinic agonists are mediated through the muscarinic M3 subtype in guinea pig ileum.