Neuropeptide Y (NPY) and its encoding mRNA were measured in neurons co-cultured from rat basal forebrain and cerebral cortex. NPY was synthesized and released in a manner consistent with secretion-synthesis coupling; depolarization increased each in a calcium-dependent manner. The accumulation of NPY encoding mRNA was elevated by a muscarinic receptor blocker, without changes in transmitter release or peptide synthesis, thereby consistent with a membrane potential-independent mechanism. Changes in intrinsic muscarinic transmission could nonetheless be expressed rapidly as an elevation in NPY levels by depolarizing the neurons subsequent to muscarinic receptor blockade. This depolarization-induced elevation of NPY subsequent to muscarinic receptor blockade was dependent on the presence of extracellular calcium ions. Forskolin and pertussis toxin also increased NPY encoding mRNA levels in a manner that was not additive with muscarinic receptor blockade. These results suggest that one or more muscarinic receptors may tonically modulate NPY synthesis via changes in adenylate cyclase activity, providing a model for the non-homeostatic modulation of neuropeptide turnover.