The alpha adrenergic receptor subtypes of the human prostate have been intensively investigated, while the muscarinic receptor subtypes and their function have yet to be determined in this tissue. [3H]-QNB binding to muscarinic receptors was performed on membrane homogenates of adenoma from six prostatectomy specimens resulting in an average total receptor density of 46 fMol/mg protein. Pirenzepine, hexahydrosiladifenidol, and para-fluoro-hexahydrosiladifenidol, drugs with high affinity for the M1 subtype, were significantly more potent inhibitors of [3H]-QNB binding than the M2 selective drug methoctramine. Immunoprecipitation studies were done using antisera raised to individual M1-M5 receptor subtypes. Approximately 75% of the solubilized receptors in the adenoma specimens were immunoprecipitated with the anti-M1 antibody, in contrast to 5% or less with antibodies against M2, M3 or M4 subtypes. These immunoprecipitation studies confirm the preponderance of the M1 subtype in prostate adenoma suggested by the high affinity pirenzepine binding. M1 receptors, when incubated with agonist, coimmunoprecipitated with the alpha subunits of the guanine nucleotide binding regulatory proteins Gi alpha, Gq/11 alpha and G16 alpha. Immunohistochemical staining with the anti-M1 antibody demonstrates the M1 receptor to be localized to the glandular epithelium. The human prostate is the first peripheral tissue in which a preponderance of the M1 subtype of muscarinic receptors has been demonstrated.