A 2,200 base pair (bp) fragment containing the 5'-flanking region of the mouse DNA polymerase beta gene was placed adjacent to and upstream of the chloramphenicol acetyl transferase (CAT)-coding region of the CAT vector. A transient expression assay of CAT activity in mouse NIH/3T3 cells transfected with this recombinant plasmid or a set of its 5'-deletion derivatives was carried out to identify a cis-acting regulatory element(s) for DNA polymerase beta gene expression. Depending on the extent of the deletion, CAT activity was dramatically increased, indicating the existence of a negative regulatory region which could be divided into two distinct domains: removal of the first domain (NRE-I), nucleotides -1860 to -1580 (+1 denotes the position of 5'-most proximal transcription initiation site), caused two to three-fold stimulation of CAT activity, and removal of the second domain (NRE-II), nucleotides -828 to -456, stimulated CAT expression another two to three-fold. When an 1,864-bp segment containing these negative regulatory elements (-2190 to -327) was inserted in the plasmid carrying the simian virus 40 early promoter and enhancer-directed CAT gene, it inhibited the CAT expression relatively independently of the orientation of insertion and the distance from the promoter-enhancer. We also mapped the promoter element of the DNA polymerase beta gene to within a 133-bp DNA fragment from nucleotide position -100 to +33. Either the NRE-I region or the NRE-II region alone can inhibit DNA polymerase beta gene promoter function.