Hydrolsis of a-naphtyl palmitate dispersed with the detergent Triton X-100 at acid pH was studied by a histochemical diazocoupling technique in both fixed sections and cultures of primate tissues as well as by a biochemical assay employing the same chromogenic substrate. Evidence for the exclusive hydrolysis of this artificial fatty acid ester substrate by acid lipases was gathered from (1) comparison of isoelectric focusing zymograms developed with different substrates, (2) kinetic analysis of enzyme activity in the presence or absence of inhibitors, including a natural substrate of acid lipase, trioleylglycerol, (3) specific localization of marked enzyme activity in certain tissues, and (4) absence of detectable enzyme activity in a case of human acid lipase deficiency (Wolman's disease). Histochemically, acid lipase activity was most readily detected in cells active in the uptake and processing of neutral lipids, i.e., the phagocytes of the reticuloendothelial system, the adrenal cortex and the lipid-storing cells in the athero-sclerotic plaques of arteries.