The cholinergic innervation of the hippocampal formation of Macaca fascicularis (cynomolgus) and Macaca mulatta (rhesus) monkeys was investigated by immunohistochemical procedures using a monoclonal antibody directed against choline acetyltransferase. The distribution of choline acetyltransferase in the monkey demonstrated both similarities and differences with the staining patterns observed in the rat or with acetylcholinesterase in the monkey. While both of these latter preparations demonstrated labeled cells, for example, no choline acetyltransferase labeled neurons were observed in the monkey hippocampal formation. Choline acetyltransferase activity was restricted to fibers which varied in thickness and number of varicosities and in their regional and laminar distribution. The highest densities of labeled fibers were observed in the uncal portion of the hippocampus, in the parasubiculum, and in the entorhinal cortex; the lowest densities of labeled fibers were observed in CA1 and in midrostrocaudal levels of the dentate gyrus. In the dentate gyrus, immunoreactive fibers were densely distributed in the molecular layer and in an infragranular plexus. One of the few striking noticeable interspecies differences was observed in the dentate gyrus. In the rhesus monkey, labeled fibers in the molecular layer were divided into a superficial denser and an inner lighter lamina, whereas in M. fascicularis, the cholinergic fibers were distributed more homogeneously throughout the molecular layer. In the hippocampus proper, there was a progressive decrease in the density of ChAT-immunoreactive fibers from CA3/CA2 into CA1. The subiculum also demonstrated modest labeling which was nonetheless higher than in CA1; the border of these fields demonstrated increased fiber labeling. The density of choline acetyltransferase staining was high in the presubiculum and parasubiculum. In the entorhinal cortex, a relatively clear boundary was observed between the more heavily stained superficial layers (I, II, and III) and the more weakly labeled deep layers (V and VI), especially in the intermediate and caudal fields. A transverse decreasing gradient was observed with the densest plexus of cholinergic fibers found in the medially situated olfactory field of the entorhinal cortex and the lowest density in the laterally located caudal and lateral fields.