The nucleotide sequence of the region (total, 2,512 base pairs [bp]) from intron 2 to the 5'-flanking region was determined for the mouse DNA polymerase beta genomic clone, and the 300-bp region from intron 1 to the 5'-flanking region was also sequenced for the rat clone. At 51 bp upstream from the ATG codon which was previously suggested to be the translation initiation codon for the rat cDNA sequence, we found another ATG in the same reading frame in both mouse and rat genes. Three major transcription initiation sites (cap sites) each for rat and mouse DNA polymerase beta mRNAs were localized precisely by primer extension analysis at 51, 41, and 0 bp upstream from the first ATG codon, suggesting that this codon is used for translation initiation. The 400-bp region around exon 1 was extremely G + C rich (about 70%). Although neither a TATA box nor a CAAT box was found within the 500-bp region upstream of the 5'-most cap site, triple repeats of 5'-CCGCCC were found within the 100-bp region flanking the cap site.