Backmutations of an ochre (UAA) nonsense defect in the tyrA gene of Escherichia coli were induced by N-ethyl-N-nitrosourea (ENU) in both UmuC+ and UmuC- strains. This site is particularly flexible to base substitution mutation and all but one (TAA-->TGA) substitution can be recovered using a reversion assay. Employing direct sequencing of polymerase chain reaction-amplified genomic DNA and/or colony-hybridization methods, the changes induced by several separate doses of ENU in a total of 587 independent backmutations were investigated. In the UmuC+ strain, all possible single-base substitutions were recovered. Different frequencies for individual single-base substitutions were obtained and correlations with the surrounding base sequence could be seen. Transitions occurred most frequently at thymine residues having a purine on the 5'-side while transversions occurred more frequently at thymine residues having a cytosine on the 5'-side. In the UmuC- strain, ENU failed to induce A:T-->T:A and A:T-->C:G transversions and the frequency of A:T-->G:C transitions was reduced. However, an unidentified class of extragenic mutations were induced to a greater extent. These results suggest distinct pathways for ENU-induced mutagenesis at ethylated thymine residues and delineate several separate functions for the UmuC protein.