1. The action of hexamethonium has been studied at a range of muscarinic receptors in vitro by use of both functional and radioligand binding studies. 2. In functional studies, hexamethonium exhibited little or no significant (P less than 0.05) antagonism of contractile responses to carbachol at muscarinic receptors in the guinea-pig ileum, oesophageal muscularis mucosae, urinary bladder and trachea. However, antagonism was observed at muscarinic receptors in the guinea-pig left atria mediating negative inotropic responses and the calculated pKB value was 3.80. Hexamethonium also antagonized contractile responses to carbachol in the canine saphenous vein. The pKB value at these receptors was 3.75. 3. In the presence of 3.2 mM hexamethonium, the pA2 value for methoctramine at atrial muscarinic receptors was reduced by approximately 10 fold (control pA2 value was 7.81 +/- 0.05; pA2 value in hexamethonium was 6.73 +/- 0.04). In contrast at tracheal muscarinic receptors, the pA2 values for methoctramine were unaffected in the presence of 3.2 mM hexamethonium (control pA2 = 5.58 +/- 0.07; pA2 value in hexamethonium was 5.63 +/- 0.12). All values quoted are mean +/- s.e. mean, n = 8. 4. In competition radioligand binding studies, hexamethonium exhibited a higher affinity for cardiac M2 receptors (pKi = 3.68) than for cerebrocortical M1 receptors (pKi = 3.28) or for submaxillary gland M3 receptors (pKi = 2.61). At M2 receptors hexamethonium at concentrations of 0.1-10 mM, increased the half life of the dissociation rate of [3H]-N-methylscopolamine 1.6-4.3 fold. This was observed at M3 receptors only at 10 mM, when the half life was increased 1.7 fold. 5. We conclude that hexamethonium, in addition to its well characterized nicotinic antagonist properties, can act as a weak muscarinic antagonist and differentiates between cardiac M2 receptors and glandular/smooth muscle M3 receptors. However, hexamethonium differentiates less clearly between M1 and M2 receptors. The selectivity between M2 and M3 receptors observed in the present study with hexamethonium is comparable to other M2 selective antagonists such as AF-DX 116 and himbacine. 6. Caution should be exercised with regard to the inclusion of hexamethonium in functionsal studies of M2 muscarinic receptor subtypes at concentrations of 0.1 mm and above.