Human promyelocytic leukemia HL-60 cells are differentiated into monocytic or granulocytic lineage when treated with 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] or all-trans retinoic acid, respectively. In this study, the effect of capsaicin, an active component of the red pepper of the genus Capsocum, on cell differentiation was investigated in a HL-60 cell culture system. Treatment of HL-60 cells with 5-30 microg/ml capsaicin for 72 h inhibited cell proliferation and induced a small increase in cell differentiation. Interestingly, synergistic induction of HL-60 cell differentiation was observed when capsaicin was combined with either 5 nM 1,25-(OH)2D3 or 50 nM all-trans retinoic acid. Flow cytometric analysis indicated that combinations of 1,25-(OH)2D3 and capsaicin stimulated differentiation predominantly to monocytes whereas combinations of all-trans retinoic acid and capsaicin stimulated differentiation predominantly to granulocytes. Capsaicin enhanced protein kinase C activity in 1,25-(OH)2D3- and all-trans retinoic acid-treated HL-60 cells. In addition, inhibitors for protein kinase C [bisindolylmaleimide (GF-109203X), chelerythrine, 1-(5-isoquinolinesulfonyl)-2-methylpiperazine dihydrochloride (H-7)] and an inhibitor for extracellular signal-regulated kinase [2-(2'-amino-3'-methoxyphenyl)-oxanaphthalen-4-one (PD-098059)] significantly inhibited HL-60 cell differentiation induced by capsaicin in combination with either 1,25-(OH)2D3 or all-trans retinoic acid. These results indicate that capsaicin potentiates 1,25-(OH)2D3- or all-trans retinoic acid-induced HL-60 cell differentiation and that both protein kinase C and extracellular signal-regulated kinase are involved in the cell differentiation synergistically enhanced by capsaicin.