The distribution of the major cholinergic regulatory enzyme acetylcholinesterase (AChE, EC 3.1.1.7) has been extensively studied in the human brain, but the distribution of the closely related enzyme butyrylcholinesterase (BuChE, EC 3.1.1.8) is largely unknown. Because of the importance of BuChE and AChE in Alzheimer's disease, we have studied the distribution of BuChE in the normal human amygdala and hippocampal formation and compared it with that of AChE by using histochemical techniques. In the amygdala, the distribution of BuChE differed significantly from that of AChE in that BuChE was found primarily in neurons and their dendritic processes, whereas AChE was found predominantly in the neuropil. BuChE-positive neurons were present in up to 10% of the neuronal profiles in lateral, basolateral (basal), basomedial (accessory basal), central, cortical, and medial amygdaloid nuclei. AChE was found primarily in the neuropil in these nuclei with only a few AChE-positive neurons. In the hippocampal formation, BuChE was also found in neurons and not in the neuropil, whereas AChE was found in both neurons and in the neuropil. BuChE and AChE neurons were present in the polymorphic layer of the dentate gyrus, as well as the stratum oriens and stratum pyramidale of the hippocampus proper. There was considerable overlap in shapes, sizes, and numbers of BuChE- and AChE-positive neurons, suggesting that the enzymes were colocalized in neurons of the hippocampal formation. The distinct distribution of BuChE suggests that it may have specific functions including coregulation of cholinergic and noncholinergic neurotransmission in human amygdala and hippocampal formation.