Previously, we provided evidence for the presence of a class of muscarinic receptors on human luteinized granulosa cells (human GC) that is linked to transient increases in intracellular free calcium levels, but not to steroid production. The precise nature of the receptor is not known, and neither its function nor the source of its natural ligand acetylcholine (ACh) is clear. To address these issues we used RT-PCR approaches and isolated complementary DNAs corresponding to the M1 receptor subtype from reverse transcribed human GC messenger ribonucleic acids. M1 receptors were further shown by immunocytochemistry, using a M1 receptor antiserum. Single cell calcium measurements showed that the M1 receptor was functionally active and linked to acute increases in intracellular free calcium, as the M1 receptor specific antagonist pirenzepine blocked the Ca2+-mobilizing effect of oxotremorine M (a muscarinic agonist). An unexpected consequence of M1 receptor activation was evidenced by the ability of muscarinic agonists to stimulate the proliferation of human GC within 24 h. In vivo, ACh, the natural ligand of these receptors is thought to be contained in cholinergic nerve fibers innervating the ovary. Surprisingly, the prerequisite for the synthesis of ACh, the enzyme choline-acetyltransferase (ChAT), is also expressed by human GC, as shown by Western blotting and immunocytochemistry. In addition, these cells express another marker for ACh synthesis, namely the gene for the vesicular acetylcholine transporter, as evidenced by RT-PCR cloning, Western blotting, and immunocytochemistry. In conclusion, our data identify the M1 receptor in human GC and point to a novel, trophic role of the neurotransmitter ACh. Furthermore, the presence of the prerequisites of ACh synthesis in human GC indicate that an autocrine/paracrine regulatory loop also exists in the in vivo counterparts of these cells in the ovary, i.e. in the cells of the preovulatory follicle and/or of the young corpus luteum.