Glucocorticoids are known to influence DNA and RNA synthesis in skin fibroblasts. In a novel approach to study this effect, we investigated the influence of the hormone analogue dexamethasone on the activity of nuclear envelope-associated nucleoside triphosphatase (NTPase) in intact cell systems (3T3 fibroblasts and MMLI melanoma cells). The NTPase is thought to be responsible for regulation of nucleocytoplasmic transport of mRNA. [3H]Dexamethasone was found to bind to nuclear ghosts at a density comparable with that of nuclear pores in this cellular fraction. Incubation of the cells for 48 h in the presence of different concentrations of dexamethasone resulted in a marked decrease of NTPase activity. Already concentrations as low as 0.1 ng/ml (3T3) or 1 ng/ml (MMLI) reduced the NTPase activity by approximately 50%. These results suggest that nuclear envelope NTPase is a site at which glucocorticoids regulate gene expression.