We have established a C2-ceramide-resistant HL-60 subline (HL-CR). HL-CR cells were resistant not only to C2-ceramide but also to various anticancer drugs. HL-CR cells did not respond to differentiation-inducing reagents including 1alpha,25-dihydroxyvitamin D(3), retinoic acid, and 12-O-tetradecanoylphorbol-13-acetate (TPA). TPA induced apoptosis in HL-CR cells much slower than in parental HL-60 cells. As it was reported that PKC isozymes were involved in C2-ceramide-induced apoptosis, we investigated the role of PKC isozymes in C2-ceramide resistance in HL-CR cells. The protein level of PKC delta was lower in HL-CR cells than in parental HL-60 cells, whereas the levels of PKC alpha, betaI, epsilon, and zeta were rather higher in HL-CR cells than in parental cells. Translocation of PKC delta from membrane to cytosol was induced by C2-ceramide in HL-CR cells as well as in wild-type HL-60 cells. Furthermore, overexpression of PKC delta in HL-CR cells potentiated C2-ceramide- and TPA-induced apoptosis and growth inhibition. These results suggest a role for ceramide in apoptosis and differentiation in HL-60 cells, and also suggest that PKC delta might be involved in ceramide- and TPA-induced apoptosis.