A collection of 164 spontaneous lacI- mutations were recovered from a uracil-DNA glycosylase deficient (Ung-) strain of Escherichia coli and analyzed by DNA sequencing. As predicted by genetic studies, G:C----A:T transitions predominated among base substitution events. However, DNA sequence analysis indicated that these events did not occur at random. Of the 31 G:C----A:T transitions recovered, 24 involved cytosine residues located in the nontranscribed strand of the gene and 15 of the 31 transitions occurred at cytosines located on the 3' side of 3 or more A:T base pairs. These differentials likely reflect the more single-stranded character of the non-transcribed strand of the gene and of regions rich in A:T base pairs. In addition, mutation at the frameshift hotspot was altered in the Ung- strain, suggesting a role for DNA repair in the formation of structural intermediates that potentiate these events. Also, the analysis of non-hotspot frameshifts, deletions and duplications showed that many involved local DNA sequence. Specifically, several of the frameshift, deletion and duplication mutations occurred near the sequence 5'-CTGG-3'. Thus, DNA sequence analysis of mutational specificity in an Ung- strain has provided evidence that gene expression, DNA repair and DNA context can all potentially influence the classes and frequencies of spontaneous mutation.