A combined method of phosphatase histochemistry and (3H)thymidine radioautography was devised to study the subcellular localization of alkaline phosphatase (AP) activiity with changing pattern of cell proliferation in precancerous livers of rats fed dimethylaminoazobenzene. After 50 hr of continuous infusion of (3H)thymidine into the rats, labeled liver tissue were fixed in glutaraldehyde. Sections were incubated for AP activity in a lead citrate medium (pH 9.4) with beta-glycerophosphate as substrate. Light and electron microscopic examinations of radioautographs revealed that focal groups of 3H-labeled hepatocytes within hyperplastic nodules were coincident to hyperbasophilic foci and distinguishable from the surrounding parenchyma, which was sparsely labeled. Proliferative hepatocytes in the foci exhibited enzyme reaction product indicative of AP activity along the entire surface membranes. The surface AP tography was in contrast to that of the surrounding hyperplastic parencyma, in which regenerative hepatocytes showed a normal localization of AP activity at the bile canalicular membranes. The L-phenylalanine-sensitive snd heat-resistant activity of hyperbasophilic hepatocytes was different from that of normal hepatocytes. The surface enzyme differentiation was accompanied by a decrease of cytoplasmic AP. Golgi elements apparently function in the mobilization of AP into the surface membranes. The phenomena of AP alterations might be related to the abnormal control of cell proliferation and cytodifferentiation leading to malignant growth.