In this report we studied coupling of M₂ and M₄ muscarinic acetylcholine receptors to activation of endothelial nitric oxide synthase (eNOS). Chinese hamster ovary cells that co-express the individual receptor subtypes and eNOS in a stable fashion were used as a model. Activation of eNOS was assayed by measuring increasing levels of cyclic GMP in admixed cells that contain guanylate cyclase. Activation of both M₂ or M₄ muscarinic receptors resulted in marked activation of eNOS, in a time- and concentration-dependent manner. The time course of the response exhibited a transient peak, followed by a sustained lower plateau. While the sustained phase was dependent on influx of extracellular calcium, the transient response showed dependency on both mobilization of intracellular calcium and extracellular influx.