Glucocorticoids have a wide range of effects in mammalian tissues. In this study we investigated the hypothesis that some of the long-term effects of glucocorticoids on immune cell function may occur by regulating phospholipase C (PLC) signal transduction systems that are known to play a role in immune cell activation. PLC activity was measured in vitro with [3H]phosphatidylinositol and [3H]phosphatidylinositol 4,5-bisphosphate as substrates. Although the guanine nucleotide regulatory protein, Gi, is a membrane-associated protein, our unpublished observations show significant Gi levels in the cytosol of several tissues, including the spleen, where the highest levels were detected. We measured cytosolic Gi alpha immunoreactivity after hormone treatment to establish the relationship between the regulation of cytosolic PLC activity and cytosolic Gi alpha levels. GTP and its nonhydrolyzable analogs have been shown in some instances to regulate soluble PLC activity. In vivo administration of dexamethasone (DEX; 5 mg/ml, sc) to rats for 24 h reduced soluble PLC activity from spleen by 25-50%. In the same tissue, cytosolic Gi alpha immunoreactivity was decreased by 60-70%. The time dependency and receptor specificity of the glucocorticoid effects observed in vivo were investigated further in isolated splenocytes. Treatment of intact splenocytes with DEX (10(-8) and 10(-7) M) for 48 h inhibited calcium-stimulated cytosolic PLC activity by 80-90%; cytosolic Gi alpha immunoreactivity was also reduced by about 90%. In a time course experiment with DEX (10(-7) M) in splenocytes, significant effects were apparent by 12 h after steroid treatment and were maximal by 48 h. When splenocytes were coincubated with DEX (10(-8) M) and the glucocorticoid receptor antagonist RU 486 (10(-7) M), the effects of DEX on soluble PLC activity and cytosolic Gi alpha immunoreactivity were both blocked, suggesting that the effects were mediated by DEX activation of the classical intracellular glucocorticoid receptor. The effects of glucocorticoids reported here may represent one way by which these hormones act to modulate immune cell function.