Hemopexin, which acts as an antioxidant by binding heme (Kd < 1 pM), is synthesized by hepatic parenchymal cells, by neurons of the central and peripheral nervous systems, and by human retinal ganglia. Two key regulatory molecules, nitric oxide (.NO) and carbon monoxide (CO), both bind to heme proteins and since ferroheme-hemopexin binds CO, the possible role of heme-hemopexin in binding .NO was investigated. .NO binds rapidly to hemopexin-bound ferroheme as shown by characteristic changes in the Soret and visible-region absorbance spectra. Circular dichroism spectra of .NO-ferroheme-hemopexin in the Soret region exhibit an unusual bisignate feature with a zero crossover at the absorbance wavelength maximum, showing that exciton coupling is occurring. Notably, the .NO complex of ferroheme-hemopexin is sufficiently avid and stable to allow hemopexin to bind this molecule in vivo and, thus, hemopexin may protect against NO-mediated toxicity especially in conditions of trauma and hemolysis.