Nuclear glucocorticoid receptor number, glucocorticoid effects on protein synthesis, and concanavalin A effects on protein synthesis were examined in rat splenic and thymic lymphocytes on Days 1 to 7 following exposure to a single 300-mg/kg injection of cyclophosphamide in vivo. Significant decreases in nuclear glucocorticoid binding occurred within 1 day after cyclophosphamide treatment. Binding continues to decrease until Day 3 and recovers partially by Day 7. Glucocorticoid receptor subcellular distribution studies demonstrate that receptor binding of glucocorticoids is decreased in both cytoplasmic and nuclear subcellular compartments when whole cells are incubated at 0 and 37°, respectively. No inhibitory effects of glucocorticoids on leucine incorporation into trichloroacetic acid-precipitable material were observed in both spleen and thymic lymphocytes for at least 3 days after the initial cyclophosphamide treatment. Maximal stimulation of leucine incorporation into protein by concanavalin A was suppressed during the entire experimental time course in both tissues, although partial recovery of the response was observed in splenic lymphocytes on Day 7. Unlike thymocytes, which responded to glucocorticoids with an inhibition of protein synthesis by Day 4, spleen cells were unresponsive for at least 7 days. The magnitude of the decrease in nuclear glucocorticoid-receptor binding decreased glucocorticoid responsiveness, and response to concanavalin A was dependent on the dose of cyclophosphamide administered in vivo. Drug concentrations as low as 25 mg/kg were sufficient to reduce receptor binding, glucocorticoid effects, and mitogen effects on protein synthesis. These observations suggest that the administration of cyclophosphamide in vivo destroys the capability of remaining splenic and thymic lymphocytes to respond to glucocorticoids.