Acetylcholine (ACh) concentration, choline acetyltransferase (ChAT) and acetylcholinesterase (AChE) were measured in 60 discrete areas dissected from the rat forebrain. All 3 substances were detectable in every region examined. The range for ACh levels was approximately 9-fold, with highest levels in the striatal and mesolimbic areas. Wider ranges were found for ChAT and AChE. In addition to not having a uniform distribution ACh, ChAT and AChE did not always show proportional variations. ACh levels did not appear to relate to the activity of either enzyme in a simple manner. There was a better correlation (r = 0.902) between the activities of ChAT and AChE, with AChE activities always being higher. In some regions, AChE was disproportionately low or high relative to ChAT. In general, the biochemical results presented here are compatible with histochemical studies of AChE. Such measurements in small brain regions should prove valuable in future experiments designed to determine cholinergic function and localize cholinergic pathways.