In contrast to the apparent paucity of Mycobacterium tuberculosis response to reactive oxygen intermediates, this organism has evolved a specific response to nitric oxide challenge. Exposure of M. tuberculosis to NO donors induces the synthesis of a set of polypeptides that have been collectively termed Nox. In this work, the most prominent Nox polypeptide, Nox16, was identified by immunoblotting and by N-terminal sequencing as the alpha-crystallin-related, 16-kDa small heat shock protein, sHsp16. A panel of chemically diverse donors of nitric oxide, with the exception of nitroprusside, induced sHsp16 (Nox16). Nitroprusside, a coordination complex of Fe2+ with a nitrosonium (NO+) ion, induced a 19-kDa polypeptide (Nox19) homologous to the nonheme bacterial ferritins. We conclude that the NO response in M. tuberculosis is dominated by increased synthesis of the alpha-crystallin homolog sHsp16, previously implicated in stationary-phase processes and found in this study to be a major M. tuberculosis protein induced upon exposure to reactive nitrogen intermediates.