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 PKCα, PKCβ, PKCβI, and PKCβII 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 PKCβ antisense (30 μM) 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 PKCβ antisense 24 h after 1,25-(OH)2D3 were unaffected and fully differentiated. PKCα antisense did not block 1,25-(OH)2D3 promotion of HL-60 cell differentiation. Next, the ability of PKCβI- and PKCβII-specific antisense oligonucleotides to block 1,25-(OH)2D3 promotion of cell differentiation was examined. PKCβII antisense (30 μM) completely blocked CD14 expression induced by 1,25-(OH)2D3, whereas PKCβI antisense had little effect. Interestingly, PKCβII 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 PKCβII expression.