In response to injury, the highly specialized and terminally differentiated glomerular visceral epithelial cell, or podocyte, may undergo several cell fates, including dedifferentiation and proliferation, persistent cell cycle arrest, hypertrophy, apoptosis, or necrosis. Common to these potential outcomes of injury is their ultimate regulation at the level of the cell cycle. There is now a large body of literature confirming the importance of cell cycle regulatory proteins in the cellular response to injury. Although CDK inhibitor p21 levels increase in podocytes following injury, the role of p21 is unclear in focal segmental glomerulosclerosis (FSGS), in part because its function depends heavily on the cytotoxic stimulus and the cellular context. Adriamycin (ADR) is a podocyte toxin used to induce experimental FSGS. The purpose of this study was to define the role of p21 in ADR-induced podocyte injury. BALB/c mice, a strain carrying the recessive ADR susceptibility gene, were backcrossed against c57B6 p21-/- mice to yield a 12th generation BALB/c p21-/- strain. Experimental FSGS was induced by injection of ADR 12 mg/kg × 2 doses (n = 8/group), with mice killed at 1, 2, 8, and 11 wk. Diseased p21-/- mice demonstrated worse albuminuria, more widespread glomerulosclerosis, and higher blood urea nitrogen compared with diseased p21+/+ mice. In diseased p21-/- mice vs. p21+/+ mice, apoptosis [measured by TdT-mediated dUTP nick end labeling (TUNEL) assay] was increased, and podocyte number (measured by WT-1 immunostaining) was decreased. To validate these findings in vitro, we utilized differentiated mouse podocytes, p21-/- and p21+/+, exposed to 0.125 μg/ml ADR. Apoptosis, measured by Hoechst 33342 staining and TUNEL assay, was greater in cultured p21-/- podocytes compared with p21+/+ podocytes. Reconstitution of p21 via retroviral transfection rescued the p21-/- podocytes from apoptosis. We conclude that p21 is prosurvival in the podocyte's response to ADR-induced injury. Ongoing studies are defining the mechanisms of this protective effect as it relates to DNA damage and apoptosis.