We studied the effects of nitric oxide (NO) on the control of excess cellular heme and release of catalytically active iron. Endothelial cells (ECs) exposed to hemin followed by a NO donor have a ferritin content that is 16% that of cells exposed to hemin alone. Hemin-treated ECs experience a 3.5- fold rise in non-heme, catalytic iron 2 h later, but a hemin rechallenge 20 h later results in only a 24% increase. The addition of a NO donor after the first heroin exposure prevents this adaptive response, presumably due to effects on ferritin synthesis. NO donors were found to reduce iron release from hemin, while heroin accumulated in cells. A NO donor, in a dose- dependent fashion, inhibited heme oxygenase activity, measured by bilirubin production. Using low temperature EPR spectroscopy, heme oxygenase inhibition correlated with nitrosylation of free heme in microsomes. Nitrosylation of cellular heme prevented iron release, for while there was heme oxygenase- dependent release of iron in cells incubated with hemin for 24 h, the addition of a NO donor blocked iron release. This indicates that NO readily nitrosylates intracellular free heme and prevents its degradation by heme oxygenase. Nitrosylation of heme was found to reduce sensitization of cells to oxidative injury.