We studied the changes of protein phosphorylation in human leukemic cells by granuloid inducers, using two-dimensional electrophoresis. The phosphorylation of 22 kD, pI 6.0 and 5.8 proteins (pp22) in HL-60 cells or myeloid leukemic cells from patients, was enhanced by treatment with granuloid inducers such as retinoic acid, dimethyl sulfoxide or G-CSF, in common with prostaglandin E 2 and theophylline, or dibutyryl c-AMP, which increased intracellular c-AMP. In contrast, pp22 phosphorylation was not induced by the monocytes/macrophages inducer in HL-60 cells, or by the granuloid inducers in lymphoid cells. This phosphorylation occurred within 30 min and continued for more than 48 h. These pp22 proteins were present in the cytosol and phosphorylated on the serine residues. We now present a possibility that granuloid differentiation in myeloid cells is closely linked with these pp22 phosphorylation.