The current study was designed to test the role of cytochrome P-450 in hydrogen peroxide-induced cytotoxicity to LLC-PK1 cells, and to determine whether it may serve as a source of catalytic iron. Hydrogen peroxide led to iron release as measured by Fe2+-(BPS)3 from the microsomes prepared from LLC-PK1 cells. Cimetidine, which inhibits cytochrome P-450 by interacting with the heme iron, significantly blocked iron release, whereas ranitidine which has a similar structure as cimetidine but is a weak inhibitor of P-450, did not have an effect (H2O2: 0.42± 0.04, H2O2 + cimetidine: 0.23 ± 0.02 nmol/mg protein, n=4, p < 0.01). Exposure of LLC-PK1 cells to hydrogen peroxide (2.5 mM) resulted in a significant increase in the bleomycin-detectable iron (iron capable of catalyzing free radical reactions) content which was prevented by cimetidine, but not ranitidine. We then examined the effect of the inhibitors of cytochrome P-450 on cell death (as measured by trypan blue exclusion) after exposure of LLC-PK1 cells to 2.5 mM hydrogen peroxide for 120 min. Inhibition of cytochrome P-450 by cimetidine significantly reduced the cell death. The effect was observed with 0.05 mM and was concentration dependent with 1 mM affording almost complete protection (H2O2: 58.5 ± 1.3 vs H2O2 + cimetidine: 10.9 ± 0.7, n=5, p < 0.01). In contrast, ranitidine did not show any protection. We confirmed that the protective effect of cimetidine was not related to scavenging hydrogen peroxide or hydroxyl radicals or chelating iron. A second inhibitor of cytochrome P-450, piperonyl butoxide, had a similar dose-dependent beneficial effect against hydrogen peroxide-induced cell injury. Our data thus indicate an important role of cytochrome P-450 in hydrogen peroxide-induced cytotoxicity to LLC-PK1 cells suggesting that cytochrome P-450 may serve as a source of catalytic iron.