To evaluate mechanisms in hemoglobinuric acute renal failure (ARF) rats were infused with hemoglobin under aciduric or alkalinuric conditions. Aciduric rats developed azotemia, distal heme casts, and proximal tubular cell (PTC) necrosis, whereas alkalinuric rats developed no renal damage. Aciduria converted hemoglobin to met-hemoglobin, which precipitated, forming distal casts and inducing ARF. Hematin formation was not observed. The importance of met-hemoglobin production was indicated by its greater toxicity than hemoglobin during aciduria and by its ability to induce ARF even under alkalinuric conditions. A link between obstructing casts and PTC necrosis was identified; tubular obstruction induced by various mechanisms (met-hemoglobin casts; ureteral ligation; ischemic ARF) increased PTC hemoglobin uptake, producing lysosomal overload (giant endolysosomes) and PTC necrosis. This worsened ischemic ARF despite an otherwise subtoxic hemoglobin dose being used that had no discernible acute renal vasoconstrictive effect. Iron chelation (deferoxamine)/hydroxyl radical scavenger (Na benzoate) therapy did not mitigate this exacerbation of ischemic injury, suggesting a nonoxidant mechanism. We conclude that H is nephrotoxic, particularly when intratubular obstruction facilitates PTC heme uptake. Thus aciduria-induced met-hemoglobin cast formation and concomitant ischemic renal injury predispose to its nephrotoxic effect.