Abstract The nitration of bovine pancreatic ribonuclease with tetranitromethane has been studied. A procedure involving solvent extraction of excess reagent and product is described. A spectrophotometric procedure for determining the extent of nitration of tyrosines has been devised, which is not vitiated by the presence of other chromophoric nitration products. In aqueous media the number of tyrosines in ribonuclease nitrated does not exceed 3. The reaction shows steps indicating differential reactivity of the different tyrosine residues. At high concentrations of reagent other reactions occur which give rise to strong new absorption. The enzyme remains active throughout, and is not substantially unfolded, as judged by optical rotatory dispersion, though there is evidence of some conformational disturbance. Spectrophotometric titrations on the nitrotyrosine residues show largely normal behaviour, but with a large electrostatic interaction parameter (spreading of the ionisation curve). At low levels of nitration however, there is consistent evidence of anomalous nitrotyrosines, titrating above pH 10. This behaviour is not observed at high levels of nitration, where the conformation is somewhat disturbed, according to a number of criteria. The anomalous nitrotyrosines amount to a fraction of a residue per protein, and it is concluded that nitration of a “masked” tyrosine has occurred in some molecules only. It has not so far proved possible to separate these. At all levels of nitration the “masked”, anomalously titrating, tyrosine residues are largely unreacted, and as in the native protein, are normalised only on denaturation. Circular dichroism reveals the presence of a new Cotton effect due to nitrotyrosine which shifts to longer wavelengths on ionisation. The tyrosine Cotton effect remains, but at alkaline pH there is no longer a contribution from phenolate chromophores. This supports the contention that the extrinsic Cotton effect in native ribonuclease is associated with the buried tyrosines, and suggests that the alkaline Cotton effect arises from a titratable residue making no large contribution at neutrality.