The present study was performed to investigate involvement of protein kinase C in the biphasic effects of 12-O-tetradecanoylphorbol-13-acetate (TPA) on cell morphology in low calcium (0.07 mM)-grown cells of a human epidermal squamous cell carcinoma cell line. The low calcium-grown cells formed no desmosomal cell-cell contact and showed roughly circular arrangements of keratin intermediate filaments around the nucleus. Treatment with 10 ng/ml of TPA induced a rapid formation (within 15 min) of cell-cell contact and reorganization of keratin intermediate filaments from a circular organization to a radial arrangement in these low calcium-grown cells. These structural phenomena were associated with a transient increase in membrane-bound protein kinase C activity. However, the prolonged treatment longer than 24 h led to a prominent decrease in the number of cell-cell contacts, that had been once formed, and caused fibroblastic changes of cell morphology in association with a decrease in the membrane-bound protein kinase C activity. Addition of 20 microM 1-(5-isoquinolinesulfonyl)-2-methylpiperazine dihydrochloride, a potential inhibitor of protein kinase C, to the medium with TPA blocked the formation of cell-cell contact. Addition of 1-(5-isoquinolinesulfonyl)-2-methylpiperazine dihydrochloride alone to normal calcium-grown cell cultures exhibiting cell-cell contact resulted in a decrease in the number of cell-cell contacts and in the fibroblastic morphological changes after 24-h incubation. These results suggest that the effects of TPA are biphasic as follows: the initial stage, inducing cell-cell contact formation associated with the translocation of protein kinase C activity from the cytosol to the membrane; and the late stage, exhibiting a fibroblastic morphological change with a decrease in the number of cell-cell contacts associated with the down regulation of this enzyme activity by TPA.