Peripheral lymphopenia occurs in man after administration of glucocorticoids. By using subset-specific monoclonal antibodies, the number of peripheral T cells (OKT11+) and the number of cells in the helper-inducer (OKT4+) and suppressor-cytotoxic (OKT8+) subsets were determined in four healthy subjects before and after ingestion of 20 mg of prednisone. In all subjects, the number of circulating T cells declined. The mean ratio of OKT4+ to OKT8+ cells dropped from 2.3 to 1.6 at 4 hr after prednisone ingestion and returned to baseline at 24 hr. The changed ratio was due to a disproportionate decrease in OKT4+ cells. A smaller decrease in the absolute number of circulating OKT8+ cells occurred, but the proportion of OKT8+ cells did not change. There was a direct relationship between prednisone dose and the reduction in circulating OKT4+ cells in the one subject who took four different doses of prednisone; there was a lymphopenic effect at the 5 mg dose. In vitro exposure of lymphocytes to methylprednisolone did not result in significant lympholysis or in alteration of the relative numbers of cells in the OKT4+ and OKT8+ subsets. The lymphopenia produced by prednisone in a splenectomized healthy subject was indistinguishable from that observed in nonsplenectomized subjects. The number of circulating B cells was unaffected by 20 mg of prednisone. These studies demonstrate that prednisone-induced lymphopenia involves the OKT4+ subset to a greater degree than the OKT8+ subset. The effect could not be accounted for by lympholysis or by redistribution of lymphocytes to the spleen.