The in vivo study aimed to examine whether muscarinic receptor subtypes other than muscarinic M3 receptors exert exocrine functional roles in the rat salivary glands. The effects of pirenzepine, methoctramine and 4-diphenylacetoxy-N-methylpiperidine methiodide (4-DAMP) were examined on secretion from the major salivary glands evoked by acetylcholine (0.001-10 micromol kg(-1) i.v.) in pentobarbitone-anaesthetized rats. Observations were occasionally made on glandular blood flow. 4-DAMP (0.1-100 nmol kg(-1) i.v.) markedly and equipotently inhibited the acetylcholine-evoked fluid responses in all glands. Pirenzepine (0.1 micromol kg(-1) i.v.-10 mmol kg(-1) i.v.) showed significantly lower inhibitory potency than 4-DAMP, most conspicuously in the parotid, while methoctramine (0.1 micromol kg(-1) i.v.-10 mmol kg(-1) i.v.) exerted an even lesser inhibitory effect. Also against acetylcholine-evoked blood flow increases, 4-DAMP showed a conspicuous potency. At 1 and 10 micromol kg(-1) i.v. of pirenzepine, the antagonist reduced the protein concentration in the submandibular saliva, but not in the parotid saliva. While 4-DAMP (1 and 10 nmol kg(-1) i.v.) significantly inhibited acetylcholine-evoked protein secretory responses in the submandibular glands, methoctramine (below 10 micromol kg(-1) i.v.) affected the responses in neither gland. The reduction of the protein concentration in submandibular saliva caused by 4-DAMP and pirenzepine was inhibited by N(omega)-nitro-L-arginine methyl ester (L-NAME; 30 mg kg(-1) i.p.), while L-NAME had no or only minute effects on the parotid protein secretion. Thus, in addition to muscarinic M3 receptors, other muscarinic receptors contribute to in vivo functional responses in rat submandibular and sublingual glands. While these other receptors are muscarinic M1 receptors in the sublingual gland, they may be a different subtype, possibly muscarinic M5 receptors, in the submandibular gland. However, muscarinic M1 receptors may induce indirect effects via nitric oxide in the submandibular gland.