It is now recognized that protein kinase C (PKC) plays a critical role in 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3) promotion of HL-60 cell differentiation. In this study, the effects of phosphorothioate antisense oligonucleotides directed against PKCalpha, PKCbeta, PKCbetaI, and PKCbetaII on HL-60 promyelocyte cell differentiation and proliferation were examined. Cellular differentiation was determined by nonspecific esterase activity, nitro blue tetrazolium reduction, and CD14 surface antigen expression. Differentiation promoted by 1,25-(OH)2D3 (20 nM for 48 h) was inhibited similarly in cells treated with PKCbeta antisense (30 microM) 24 h prior to or at the same time as hormone treatment (86 +/- 9% inhibition; n = 4 versus 82 +/- 8% inhibition; n = 4 (mean +/- S.E.), respectively). In contrast, cells treated with PKCbeta antisense 24 h after 1, 25-(OH)2D3 were unaffected and fully differentiated. PKCalpha antisense did not block 1,25-(OH)2D3 promotion of HL-60 cell differentiation. Next, the ability of PKCbetaI- and PKCbetaII-specific antisense oligonucleotides to block 1,25-(OH)2D3 promotion of cell differentiation was examined. PKCbetaII antisense (30 microM) completely blocked CD14 expression induced by 1, 25-(OH)2D3, whereas PKCbetaI antisense had little effect. Interestingly, PKCbetaII antisense blocked differentiation by 87 +/- 7% (n = 2, mean +/- S.D.) but had no effect on 1,25-(OH)2D3 inhibition of cellular proliferation. These results indicate that the effects of 1,25-(OH)2D3 on HL-60 cell differentiation and proliferation can be dissociated by blocking PKCbetaII expression.