The activities of choline acetyltransferase (ChAT) and acetylcholinesterase (AChE) were measured in five neocortical regions and in the hippocampal formation of one hemisphere in eight 30-day-old squirrel monkeys. Enzyme levels in the hippocampal formation (hippocampus, dentate gyrus, and subiculum) were higher than in any neocortical region. Within neocortex, ChAT activity was highest in superior temporal and precentral regions, intermediate in prefrontal and postcentral regions, and lowest in occipital cortex. AChE activity also varied across neocortical regions and correlated significantly, but imprecisely, with ChAT activity. The activity of overall neocortical ChAT, but not AChE, differed significantly among animals. The pattern of cholinergic innervation of neocortex in the lissencephalic squirrel monkey is highly similar to that reported in gyrencephalic Old World primates.