To obtain insight into the nature and mechanisms of spontaneous mutations, Escherichia coli K12 strain TM31 was constructed to determine, by DNA sequencing, the mutational spectrum of the tonB gene on the chromosome. We inserted the chloramphenicol resistant gene 1.6 kb upstream of the tonB gene, thus making it possible to retrieve the mutated tonB gene from the chromosome by shotgun cloning using a drug-resistant marker. The spontaneous mutation frequency in the tonB gene, which was judged by its colicin B-resistant phenotype, is 3~10×10^-7. Spontaneous mutations were dominated by large insertions that are identified by DNA sequencing to be IS elements; IS1 dominated, but IS2, IS5, and IS10 were also obtained. In uvrA^- strain, transposition of both IS10-R and IS10-L are equally increased, suggesting the interaction of the UvrA protein and IS10 transposition. The base substitutions are the second largest group of mutations, among which G :C→A :T transition is predominant. Deletions also contribute significantly in wild type with regard to DNA repair and uvrA^- s trains, but not recA^- strain, suggesting that the RecA protein is involved to some extent in deletion formation. Endpoints of these deletions do not always correlate with the presence of repeated sequences, indicating the absence of homologous recombination for deletion formation.