Long-term phenylhydrazine (PHZ) treatment caused pronounced anemia and a concomitant increase in the numbers of circulating leukocytes in Long-Evans rats. The leukocytosis was caused mainly by an elevation in mononuclear cells, most notably in the lymphocyte population. PHZ has been reported to cause the direct lysis of erythrocytes by nonimmune mechanisms. However, recent reports indicate that PHZ can cross-link red cell band 3 protein (senescent antigen), resulting in the binding of autologous immunoglobulin G (IgG). Recognition of this complex by macrophage Fc receptor mechanisms triggers rapid erythrophagocytosis-in the spleen and possibly the liver as well. In our study, analysis of the blood, bone marrow, and spleen cells of long-term (1 to 6 weeks) PHZ-treated rats was performed by using flow cytometry. Total serum IgG levels were determined by radial immunodiffusion, and antibodies reactive with red cells that were sensitized to PHZ either in vivo or in vitro were titered by using the indirect Coombs' method. Serum prostaglandin E2 titers also were determined at different time intervals after PHZ administration. The results indicate that PHZ induces an increase in circulating antibody and prostaglandin E2 titers that correlates with the onset of anemia and that the serum of PHZ-treated rats can induce anemia in normal recipients after passive transfer. Cytofluorographic studies revealed a marked increase in the B-cell population of the peripheral blood and spleen and an altered ratio T-helper to T-suppressor cells at certain time intervals after PHZ injection. The results indicate that in addition to inducing senescence-like alterations in erythrocyte membrane proteins, PHZ stimulates the production of the autologous IgG that recognizes these sites and promotes lymphoid blastogenesis, most notably in the B-cell lineage.