Various concentrations of 1,25-dihydroxyvitamin D3 (vit D3; 10(-9)-10(-7) M) and recombinant human tumor necrosis factor alpha (rTNF-alpha; 60-960 U/ml) were used to induce growth inhibition and differentiation of the human promyelocytic leukemia cell line HL-60 based on growth kinetics, colony formation, morphological analysis, nonspecific esterase (NSE) activity, surface antigen expression, and cytokine release. Both vit D3 (10(-8)-10(-7) M) and rTNF-alpha (60-960 U/ml) were antiproliferative against the HL-60 cells, and a cooperative effect was noted when the two inducers were used in combination. After 5 days of incubation, vit D3 induced the HL-60 cells to differentiate into monocytes/macrophages, resulting in the formation of 3.0% +/- 0.4%, 18% +/- 2.0%, and 43% +/- 3.8% of morphologically mature cells at 10(-9), 10(-8), and 10(-7) M, respectively. The induced cells were NSE positive and expressed monocyte-associated antigens (EBM11, CD11b, and HLA-DR). Conversely, rTNF-alpha (60-960 U/ml) was unable to trigger the HL-60 cells to differentiate. However, rTNF-alpha could apparently increase the proportion of the morphologically mature and NSE-/antigen-positive cells when used in combination with vit D3 (10(-9)-10(-8) M). Following differentiation induction, HL-60 cells from vit D3-treated HL-60 cultures acquired the ability to secrete certain monokines, including interleukin 1 beta (IL-1 beta), prostaglandin E2 (PGE2), and granulocyte-macrophage colony-stimulating factor (GM-CSF), and adding rTNF-alpha in addition to vit D3 invariably increased the production of IL-1 beta and PGE2.