The human promyeloic leukemia cell line HL-60 can be triggered in culture to differentiate into several cell types of the myeloid lineage in response to a variety of chemical stimuli. We used this cell system in order to investigate the changes in glucocorticoid receptors which occur concomitantly with such cellular differentiations. Neutrophilic granulocytes obtained by the addition of dimethyl sulfoxide or retinoic acid to the culture medium showed only slight changes in cellular glucocorticoid receptor levels and receptor-specific mRNA as compared to undifferentiated control cells. Monocytic cells induced by incubation with dihydroxy-vitamin D3 had a moderate increase in receptor hormone-binding activity. However, differentiation toward macrophages by exposure to phorbol ester resulted in a 5- to 6- fold increase in both cellular hormone-binding capacity and immunochemically cross-reacting receptor protein. An even greater increase in glucocorticoid receptor-specific mRNA was observed. These data suggest that the receptor is regulated at the mRNA level and that de novo receptor synthesis occurs during macrophage differentiation, thus making these cells potentially more susceptible to glucocorticoid-induced effects.