Signaling mechanisms coupled to activation of different neurotransmitter receptors interact in the enteric nervous system. Acetylcholine (ACh) excites myenteric neurons by activating nicotinic acetylcholine receptors (nAChRs) and muscarinic receptors expressed by the same neurons. These studies tested the hypothesis that muscarinic receptor activation alters the functional properties of nAChRs in guinea pig small intestinal myenteric neurons maintained in primary culture. Whole-cell patch clamp techniques were used to measure inward currents caused by ACh (1 mM) or nicotine (1 mM). Currents caused by ACh and nicotine were blocked by hexamethonium (100 µM) and showed complete cross-desensitization. The rate and extent of nAChR desensitization was greater when recordings were obtained with ATP/GTP-containing compared to ATP/GTP-free pipette solutions. These data suggest that ATP/GTP-dependent mechanisms increase nAChR desensitization. The muscarinic receptor antagonist, scopolamine (1 µM) decreased desensitization caused by ACh but not by nicotine, which does not activate muscarinic receptors. Phorbol 12, 13 dibutyrate (10 - 100 nM), an activator of protein kinase C (PKC), but not 4--phorbol 12-myristate 13-acetate (a PKC inactive phorbol ester) increased nAChR desensitization caused by ACh and nicotine. Forskolin (1 µM), an activator of adenylate cyclase, increased nAChR desensitization but this effect was mimicked by dideoxyforskolin, an adenylate cyclase inactive forskolin analog. These data indicate that simultaneous activation of nAChRs and muscarinic receptors increases nAChR desensitization. This effect may involve activation of a PKC-dependent pathway. These data also suggest that nAChRs and muscarinic receptors are coupled functionally through an intracellular signaling pathway in myenteric neurons.