To test the hypothesis that inaccurate DNA synthesis by mammalian DNA palymerase η (pol η) contributes to somatic hypermutation (SHM) of Ig genes, we measured the error specificity of mouse pal η during synthesis of each strand of a mouse Ig κ light chain transgene. We then compared the results to the base substitution specificity of SHM of this same gene in the mouse. The in vitro and in vivo base substitution spectra shared a number of common features. A highly significant correlation was observed for overall substitutions at A-T pairs but not for substitutions at G-C pairs. Sixteen mutational hotspots at A-T pairs observed in vivo were also found in spectra generated by mouse pol η in vitro. The correlation was strongest for errors made by pal η during synthesis of the non-transcribed strand, but it was also observed for synthesis of the transcribed strand. These facts, and the distribution of substitutions generated in vivo, support the hypothesis that pol η contributes to SHM of Ig genes at A-T pairs via