To evaluate potential direct effects of glucocorticoids on the aqueous outflow pathway, the cellular binding of steroids to cultured human trabecular cells was examined. After incubation of cells with 5 to 40 nM [3H]dexamethasone, specific binding (i.e., binding that could be blocked by an excess of nonlabeled steroid) was detected by measuring the total cell-associated labeled hormone. A binding affinity of 5 nM and 60,000 receptor sites/cell were demonstrated with labeled dexamethasone. Incubation of human trabecular cells with 40 nM [3H]dexamethasone for 60 min revealed that 62% +/- 7 of the specific binding was found in the nuclear fraction and 38% +/- 3 was in the cytoplasmic fraction. In competition studies, dexamethasone had a higher affinity for these sites than cortisol, which in turn had a higher affinity than progesterone. These studies suggest that functional glucocorticoid receptors are present in human trabecular cell cultures. Therefore it is possible that a direct action of glucocorticoids on trabecular cells could contribute to the decreased outflow facility observed in steroid glaucoma.