A hydrogen peroxide-resistant mutant of the catalase-negative microaerophile, Spirillum volutans, constitutively expresses a 21.5 kDa protein that is undetectable and non-inducible in the wild-type cells. Part of the gene that encodes the protein was cloned using amino acid sequence data obtained by both mass spectrometry and NH2-terminal sequencing. The deduced 158 amino acid polypeptide shows high relatedness to rubrerythrin and nigerythrin previously described in the anaerobes Clostridium perfringens and Desulfovibrio vulgaris. The protein also shows high similarity to putative rubrerythrin proteins found in the anaerobic archeons Archaeoglobus fulgidus, Methanococcus jannaschii and Methanobacterium thermoautotrophicum. This is the first report of this type of protein in an organism that must respire with oxygen. It seems likely that the novel combination of methodologies used in this study could be applied to the rapid cloning of other genes in bacteria for which no genomic library yet exists.