A system of small to medium size choline acetyltransferase (ChAT)-containing neurons has been identified in rat, monkey and human hypothalamus. A highly sensitive polyclonal anti-human placental ChAT rabbit serum, combined with a nickel ammonium sulfate second antibody intensification method, was used to identify these relatively weakly staining ChAT-positive neurons. The most prominent hypothalamic group consisted of small neurons in the infundibular (arcuate) nucleus. Fibers extended towards the infundibulum. Other ChAT-positive cells were not identified with specific hypothalamic nuclei but were scattered loosely in the surrounding matrix. They fell into two broad complexes: a medially distributed one close to the third ventricle and running rostrocaudal to caudoventral; and a lateral one distributed principally in the region of the medial forebrain bundle. The most laterally placed hypothalamic ChAT-positive neurons slightly overlapped with the large, intensely staining cells of the medial basal forebrain cholinergic complex. The identification of these cells helps to account for previous biochemical and pharmacological studies which have strongly indicated the presence of intrinsic cholinergic neurons in the hypothalamus.