Protein kinase C (PKC) isoforms play pivotal roles in the regulation of differentiation of normal human epidermal keratinocytes (NHEK). In this study, we investigated the participation of the PKC system in the proliferation and high cell density-induced differentiation of the human immortalized keratinocyte line HaCaT. HaCaT keratinocytes possessed a characteristic PKC isoform pattern (PKC alpha, beta, gamma, delta, epsilon, eta, theta, zeta), which altered during proliferation and differentiation. The GF109203X compound, a selective PKC inhibitor, suppressed the expressions of the lat (granular cell) differentiation markers involucrin (INV) and filaggrin (FIL), and the terminal marker keratinocyte-specific transglutaminase-1 (TG), but did not affect the level of the early (spinous cell) marker keratin 10 (K10) and cellular proliferation. Phorbol 12-myristate 13-acetate (PMA), an activator of PKC, inhibited proliferation, elevated intracellular calcium concentration, decreased the expression of K10, and increased the expressions of INV, FIL, and TG. These data indicate that the endogenous activation of PKC regulates the expressions of the late differentiation markers, and that the exogenous activation of PKC by PMA results in the induction of terminal differentiation. Because the cellular effects of PMA were accompanied by differential down-regulations of the sensitive PKC isoforms in proliferating and differentiating cultures, our findings argue for the differential roles of the existing PKC isoforms in the regulation of cellular proliferation and high cell density-induced differentiation of HaCaT cells.