Cisplatin is an important chemotherapeutic agent but can cause acute renal injury. Part of this acute renal injury is mediated through tumor necrosis factor-α (TNF-α). The pathway through which cisplatin mediates the production of TNF-α and injury is not known. Cisplatin activates p38 MAPK and induces apoptosis in cancer cells. p38 MAPK activation leads to increased production of TNF-α in ischemic injury and in macrophages. However, little is known concerning the role of p38 MAPK in cisplatin-induced renal injury. Therefore, we examined the effect of cisplatin on p38 MAPK activity and the role of p38 MAPK in mediating cisplatin-induced TNF-α production and renal injury. In vitro, cisplatin caused a dose-dependent activation of p38 MAPK in proximal tubule cells. Inhibition of p38 MAPK activation led to inhibition of TNF-α production. In vivo, mice treated with a single dose of cisplatin (20 mg/kg body wt) developed severe renal dysfunction at 72 h [blood urea nitrogen (BUN): 154 ± 34 mg/dl, creatinine: 1.4 ± 0.4 mg/dl], which was accompanied by an increase in kidney p38 MAPK activity and an increase in infiltrating leukocytes. However, animals treated with the p38 MAPK inhibitor SKF-86002 along with cisplatin showed less renal dysfunction (BUN: 55 ± 14 mg/dl, creatinine: 0.3 ± 0.02 mg/dl, P < 0.05), less severe histological damage, and fewer leukocytes compared with cisplatin+vehicle-treated animals. Serum levels of TNF-α, sTNFRI, and sTNFRII also increased significantly in cisplatin-treated mice compared with SKF-86002-treated mice (P < 0.05). Kidney mRNA levels of TNF-α were significantly increased in cisplatin-treated mice compared with either SKF-86002- or saline-treated animals. The hydroxyl radical scavenger DMTU (100 mg·kg body wt⁻¹·day⁻¹) prevented the activation of p38 MAPK by cisplatin both in vitro and in vivo. DMTU also completely prevented cisplatin-induced renal injury (BUN: 140 ± 27 vs. 22 ± 2 mg/dl, P < 0.005) and the increase in serum TNF-α (33 ± 7 vs. 4 ± 2 pg/ml, P < 0.005) and kidney TNF-α mRNA in vivo. We conclude that hydroxyl radicals, either directly or indirectly, activate p38 MAPK and that p38 MAPK plays an important role in mediating cisplatin-induced acute renal injury and inflammation, perhaps through production of TNF-α.