We have examined the effect of physiologic concentrations of dexamethasone on the induction of differentiation of the human promyelocytic leukemia cells (HL-60) and the activity of surface receptors for the f-met-leu-phe chemotactic peptide. Dexamethasone at a concentration of 500 nM was markedly synergistic with dimethylformamide and dimethylsulfoxide, but not with trans-retinoic acid, in inducing f-met-leu-phe binding by HL-60 cells. The increase in peptide binding was due to an increase in the activity of peptide receptors rather than an increase in receptor affinity. The effect of dexamethasone was relatively specific for peptide binding as there was little effect when HL-60 differentiation was assessed by cell morphology or the ability of the cells to reduce nitroblue tetrazolium. Dexamethasone alone had no reproducible effect on HL-60 differentiation as assessed by the three criteria tested. The dexamethasone concentration required for half maximal synergism correlated with the known binding constant of the hormone for steroid receptors in HL-60 cells but the effect of dexamethasone was not blocked with the steroid blocking agents 17α methyltestosterone or progesterone.