Protein kinase C (PKC) is a family of serine/threonine protein kinases involved in many cellular responses. Although the analysis of PKC activity in many systems has provided crucial insights to its biologic function, the precise role of different isoforms on the differentiation of normal hematopoietic progenitor cells into the various lineages remains to be investigated. The authors have assessed the state of activation and protein expression of PKC isoforms after cytokine stimulation of CD34(+) progenitor cells from human bone marrow. Freshly isolated CD34(+) cells were found to express PKC-alpha, PKC-beta2, and PKC-epsilon, whereas PKC-delta, PKC-gamma, and PKC-zeta were not detected. Treatment with erythropoietin (EPO) or with EPO and stem cell factor (SCF) induced a predominantly erythroid differentiation of CD34(+) cells that was accompanied by the up-regulation of PKC-alpha and PKC-beta2 protein levels (11.8- and 2.5-fold, respectively) compared with cells cultured in medium. Stimulation with EPO also resulted in the nuclear translocation of PKC-alpha and PKC-beta2 isoforms. Notably, none of the PKC isoforms tested were detectable in CD34(+) cells induced to myeloid differentiation by G-CSF and SCF stimulation. The PKC inhibitors staurosporine and calphostin C prevented EPO-induced erythroid differentiation. Down-regulation of the PKC-alpha, PKC-beta2, and PKC-epsilon expression by TPA pretreatment, or the down-regulation of PKC-alpha with a specific ribozyme, also inhibited the EPO-induced erythroid differentiation of CD34(+) cells. No effect was seen with PKC-beta2-specific ribozymes. Taken together, these findings point to a novel role for the PKC-alpha isoform in mediating EPO-induced erythroid differentiation of the CD34(+) progenitor cells from human bone marrow. (Blood. 2000;95:510-518)