In this study, we characterized the pharmacology and physiology of the automodulation of ACh release at the lizard neuromuscular junction (NMJ). The activation of muscarinic ACh receptors generated a biphasic modulation of synaptic transmission. Muscarine-induced activation of M3 receptors (0–12 min) decreased release, whereas M1 activation (> 12 min) enhanced release. Both phases of the biphasic effect are dependent on nitric oxide. However, cAMP acting via protein kinase A is also necessary for the M1 effect. In summary, we present a novel biphasic role for muscarine and implicate M3 receptors in the inhibition and M1 receptors in the enhancement of transmitter releaseat the cholinergic lizard NMJ.