Human promyelocytic leukemia cells (HL-60) were treated with several differentiation inducers, then the changes in the activity of cytosolic protein kinase C (PKC) isoforms were examined by hydroxylapatite chromatography and the species of the isoforms were determined immunologically. In three undifferentiated HL-60 cell lines examined, PKC alpha and beta isoforms were present, but PKC gamma isoform was not detected. When the cells were induced by dimethylsulfoxide, dibutyryl cAMP, or nicotinamide to differentiate into granulocytes, these two PKC isoforms each increased to about 2- to 3-fold. When retinoic acid was used as the inducer, in addition to PKC alpha and beta, a third PKC isoform appeared. This isoform was clearly distinct from rat PKC alpha, beta, and gamma, immunologically. This isoform showed a distinctly lower Ca(2+)-requirement (3 microM) than that of PKC alpha or beta (100 microM) and was more dependent on cardiolipin and phosphatidylethanolamine, compared with PKC alpha, beta, and gamma. These results suggest that while the increases in the activities of PKC alpha and beta isoforms are common in the differentiation program initiated by several inducers, including retinoic acid, the emergence of an unclassified PKC isoform is a retinoic acid-specific process.