Human THP-1 leukemia cells differentiate along the monocytic lineage following exposure to phorbol-12-myristate-13-acetate (PMA) or 1,25-dihydroxyvitamin D3 (VD3). In the monocytic cell line THP-1, PMA treatment resulted in a more differentiated phenotype than VD3, according to adherence, loss of proliferation, phagocytosis of latex beads, and expression of GD11b and CD14. Both differentiating substances induced similar effects in the release of superoxide anions (O₂^-). VD₃-differentiated cells did not release prostaglandin E₂ (PGE₂), in contrast to PMA-differentiated cells, and in PMA-differentiated cells phospholipase A₂ (PLA₂) activity and expression was increased. Lipopolysaccharide (LPS)-stimulated tumor necrosis factor-α (TNF-α) release was higher in PMA-treated cells. PMA- but not VD3-differentiation resulted in a translocation of protein kinase C (PKC) isoenzymes to membrane fractions. Both differentiating agents up-regulated the expression of PKC isoenzymes. Whereas VD3 elevated mainly the expression of PKC-β, PMA caused a strong increase in PKC-δ and a weak increase in PKC-α, PKC-, and PKC- expression. These results indicate that phorbol ester and the active metabolite of vitamin D induce different signal pathways, which might result in different achievement of differentiation.