Using either hexazotized pararosaniline or new fuchsin as coupling agents, we investigated the ultrastructural localization of alpha-naphthyl acetate esterase (ANAE) activity in guinea pig bone marrow and peritoneal exudates, and in human peripheral blood cells. DFP-inhibitable ANAE activity was present on the cell surface of lymphocytes, monocytes, macrophages, neutrophils, eosinophils, basophils, megakaryocytes, platelets, and blasts. Demarcation lines in megakaryocytes and the perinuclear cisternae in normoblasts were also positive. In addition, lymphocytes, monocytes, and macrophages displayed ANAE activity associated with cytoplasmic-vesicle clusters (CVC). Reaction product was always present on the cytoplasmic surfaces of these vesicles and in the adjacent cytoplasm; vesicle interiors were invariably ANAE-negative. Small lymphocytes generally had a single large paranuclear ANAE-positive CVC, whereas mononuclear phagocytes had multiple discrete foci of similar appearing ANAE-positive CVC that sometime became confluent. ANAE activity was also found in the Gall bodies of human lymphocytes and in coated vesicles of macrophages. Cytoplasmic ANAE activity was increased in oil-induced guinea pig peritoneal macrophages. Both surface and cytoplasmic esterase activities had a neutral pH optimum. An identical distribution of reaction product was observed when alpha-naphthyl butyrate was employed as substrate. The function of these esterases, and their relation to known surface and cytoplasmic neutral proteases, awaits further investigation.