To evaluate the molecular basis for susceptibility of the cell differentiation induced by 12-O-tetradecanoylphorbol-13-acetate (TPA), we examined biochemical activities and expression of subspecies of protein kinase C from HL-60 cells that are susceptible to differentiation induced by TPA and HL-60R cells, HL-60 variant cells that are resistant to such induction. Analysis of the subcellular distribution of protein kinase C revealed that the activity of this kinase in the cytosol from HL-60R cells was 30% of that from parental HL-60 cells but that the enzyme activities in the membrane showed similar values in these cells. Treatment of HL-60 cells with 100 nM TPA for 30 min resulted in a 75% decrease in protein kinase C activity in the cytosol and a 300% increase in this activity in the membrane. A minor subcellular redistribution of the enzyme activity was found in HL-60R cells after TPA treatment. Based on analysis of protein kinase C isozymes by hydroxyapatite column chromatography, the relative activities of types I, II, and III in the cytosol of HL-60 cells were 11, 80, and 9%, whereas those in HL-60R cells were 27, 36, and 37%, respectively. Type II isozyme was a major protein kinase C in the cytosol of HL-60 cells, but type II was less in the HL-60R cells. Among the three isozymes, type II enzyme was most sensitive to TPA with histone H1 as the substrate, although all three isozymes were activated Ca2+-dependently in the presence of phosphatidylserine. We suggest that the acquired resistance of HL-60R cells toward induction of cell differentiation by TPA may be associated with a decrease in the expression of the type II isozyme of protein kinase C.