The pharmacological profile of the muscarinic stimulation of adenylate cyclase activity in rat olfactory bulb was investigated by using a number of antagonists with different selectivity for the muscarinic receptor subtypes. Schild analysis showed that this response was counteracted with the following rank order of potency: R(-)quinuclidinyl benzilate greater than atropine greater than RS(+/-)-3-quinuclidinyl xanthene-9-carboxylate hemioxalate greater than S(+)quinuclidinyl benzylate greater than 4-diphenylacetoxy-N-methylpiperidine methbromide greater than secoverine greater than methoctramine greater than dicyclomine greater than hexahydrosiladifenidol greater than p-fluorohexahydro-sila-difenidol greater than AF-DX 116 greater than pirenzepine. In the heart and corpus striatum, the antagonist rank orders of potency in counteracting the muscarinic inhibition of adenylate cyclase activity were consistent with the involvement of homogeneous populations of M2 and M3 receptors, respectively. Conversely, in the olfactory bulb, the pirenzepine inhibition curve of acetylcholine-stimulated enzyme activity was biphasic, with a major component of the response being inhibited with low affinity. These results indicate that more than one receptor subtype may participate in the stimulation of adenylate cyclase in rat olfactory bulb. The predominant receptor involved appears to be different from the M1, M2 and M3 subtypes and pharmacologically equivalent to the m4 gene product.