The mechanism by which glucocorticosteroids (GCS) suppress proliferation of human peripheral blood mononuclear leukocytes (PBML) was investigated. Using the proliferative responses to immobilized anti-CD3 mAb or mitogens (PHA + PMA) as biological readouts, dexamethasone (DEX) and 6 alpha-methylprednisolone (6 alpha-MP) were shown to inhibit PBML proliferation in a concentration-dependent fashion. The mechanism by which GCS mediate immunosuppression did not involve interference with Ca2+ fluxes as: (1) DEX failed to block Ca2+ entry into anti-CD3 + PMA stimulated cells; and (2) Ca2+ ionophores (ionomycin and A23187) failed to circumfent DEX-mediated suppression. DEX also had no effect on protein kinase C (PKC) activity as: (1) inhibitors (H-7 and staurosporin) or stimulators (1,2-dihexanoyl-sn-glycerol [DiC6] and 1,2-dioctanoyl-rac-glycerol [DiC8]) of PKC did not prevent DEX-mediated suppression; (2) DEX did not affect the activation-induced upregulation of CD4 and CD8 expression, an indirect index of PKC activity; and (3) DEX did not alter the activation-associated translocation of PKC from cytosolic to membrane-bound compartments. This, in addition to previous results demonstrating that GCS directly inhibit cytokine gene transcription and that rII-1 + rIL-6 + rIFN-gamma completely abrogated GCS-mediated suppressive effects, further supports the notion that GCS exert their immunosuppressive effects through inhibition of cytokine gene expression.