The effect of dexamethasone on the HL-60 human promyelocytic leukemia cell line was studied using a radioligand-binding assay for a chemotactic peptide receptor that is found on the surface of mature polymorphonuclear leukocytes. When dimethyl sulfoxide (DMSO) is added to cultures, HL-60 cells undergo morphological and functional differentiation over a period of 6 to 7 days. Differentiation is accompanied by increased binding of the synthetic N-formylated chemotactic peptide N-formylmethionylleucyl[3H]phenylalanine. Binding of N-formylmethionylleucyl[3H]phenylalanine to cells induced to differentiate with DMSO was temperature dependent, specific, saturable, of high affinity, reversible, and proportional to cell number. Dexamethasone increased the number of N-formylated chemotactic peptide receptors in cultures of differentiating HL-60 cells without affecting the affinity of the receptors for the tritiated peptide. This steroid response was dose dependent, proportional in magnitude to glucocorticoid activity, and abolished by cycloheximide. Dexamethasone had little effect on N-formylmethionylleucyl[3H]phenylalanine binding unless DMSO was also present in culture or the cells were first induced by DMSO to differentiate. These results suggest that the glucocorticoid stimulated increase in number of N-formylated chemotactic peptide receptors was mediated by the high-affinity glucocorticoid receptor, involved protein synthesis, and was apparently dependent on differentiation.