Cholinergic neurons of the nucleus basalis of Meynert project to numerous regions of the cerebral cortex. However, little is known about the regional and laminar distributions of cholinergic axons in monkey frontal cortex. In this study, immunohistochemical techniques were used to identify axons that were immunoreactive for choline acetyltransferase, the enzyme that catalyses the synthesis of acetylcholine, in the frontal cortex of cynomolgus monkeys. Motor cortex contained the greatest density of labeled fibers: the density of labeled fibers was lower in premotor and anterior cingulate cortices and lower still in the association regions of prefrontal cortex. On a laminar basis, choline acetyltransferase-immunoreactive axons were most dense in layer I to superficial layer III. Layer V also contained a distinct band of labeled fibers that was particularly prominent in the agranular regions of frontal cortex. The density of labeled fibers was much lower in the deep portion of layer III to layer IV and in layer VI. These findings demonstrate a specific and regionally distinctive cholinergic innervation of monkey frontal cortex that may reveal the anatomical basis for the influence of acetylcholine on the diverse functions of primate frontal cortex.