Activation of the p38 mitogen-activated protein kinase (MAPK) pathway induces inflammation, apoptosis and fibrosis. However, little is known of the contribution of the upstream kinases, MMK3 and MKK6, to activation of the p38 kinase in the kidney and consequent renal injury. This study investigated the contribution of MKK3 to p38 MAPK activation and renal injury in the obstructed kidney. Groups of 8 wild type (WT) or Mkk3-/- mice underwent unilateral ureteric obstruction (UUO) and were killed 3 or 7 days later. Western blotting showed a marked increase in phospho-p38 (p-p38) MAPK in UUO WT kidney. The same trend of increased p-p38 MAPK was seen in UUO Mkk3-/- kidney, although the actual level of p-p38 MAPK was significantly reduced compared to WT and this could not be entirely compensated for by the increase in MKK6 expression in the Mkk3-/- kidney. Apoptosis of tubular and interstitial cells in WT UUO was reduced by 50% in Mkk3-/- UUO mice. Furthermore, cultured Mkk3-/- tubular epithelial cells showed resistance to H₂O₂-induced apoptosis, suggesting a direct role for MKK3-p38 signaling in tubular apoptosis. Up-regulation of MCP-1 mRNA levels and macrophage infiltration seen on day 3 of WT UUO was significantly reduced in Mkk3-/- mice, but this difference was not evident by day 7. The development of renal fibrosis in Mkk3-/- UUO mice was not different to that seen in WT UUO mice. In conclusion, these studies identify discrete roles for MKK3-p38 signaling in renal cell apoptosis and the early inflammatory response in the obstructed kidney.