The reaction of mammalian cytochrome P-450 2B4 with nitrogen monoxide and oxygen has been studied by surface-enhanced resonance Raman scattering (SERRS) to obtain sharp and definitive information in situ on the nature of the changes in the active site pocket. The initial reaction produces a six co-ordinate low spin haem-nitrogen monoxide adduct. A slower reaction leads to the irreversible formation of a five co-ordinate high-spin iron (III) haem with no nitrogen monoxide bound to it and to the nitration of an aromatic side chain, probably a tyrosine, in the proximity of the active site. In the presence of excess nitrogen monoxide, the second reaction is controlled by oxygen concentration. The sequence of events corresponds to the biphasic inhibition induced by NO in other cytochromes P-450 and peroxidases and is postulated to occur by the formation of a nitrating agent at the haem followed by diffusion to the tyrosine. The nitrated amino acid and the oxidation and spin state of the haem are observed easily by SERRS with low concentrations of protein making it a particularly suitable method for the investigation of reactions of NO in complex biological matrices.