The distribution and morphologic characteristics of choline acetyltransferase (ChAT)-containing neurons were studied throughout the rostrocaudal extent of the rat hippocampus and in a midline area just dorsal to the dorsal hippocampus. Peroxidase reaction product was observed with the aid of immunohistochemical methods and a high-titer polyclonal antibody against ChAT, the acetylcholine biosynthetic enzyme. ChAT-positive cells in the hippocampus were characterized by small, round or oval perikarya with two or more proximal processes. They were located within the caudal and temporal hippocampal formation, predominantly within the subiculum, in the stratum lacunosum moleculare, at the border of the stratum lacunosum moleculare and the stratum radiatum, and in the molecular layer of the dentate gyrus. The cells resembled in morphology the small, bipolar and multipolar neocortical ChAT-immunoreactive cells. In addition to the hippocampus, ChAT-positive neurons were observed caudally in a region just above the dorsal hippocampal commissure and rostrally in the columns of the fornix. These cells were large with an oval perikarya and darkly labeled compared to neurons in the hippocampus. They more closely resembled the ChAT-positive neurons in the midline raphe of the medial septal nucleus. Examination of the rat hippocampus 2 and 8 weeks following unilateral lesioning of the fimbria-fornix and supracallosal striae revealed a sparse innervation of ChAT-positive fibers in the hippocampus ipsilateral to the lesion. ChAT-labeled neurons in the hippocampus did not appear to sprout in response to the lesion. In contrast, ChAT-positive cells in the midline did appear to sprout into the medial dorsal subiculum and dorsal medial hippocampus. We conclude that these two populations of cells are distinct with respect to their response to hippocampal denervation and, furthermore, that this distinction may be attributed to a differential response to nerve growth factor.