A cDNA molecule (B52) with the characteristics of retroviral sequences was isolated from the Chinese hamster ovary (CHO) K1 cell line. B52 cDNA detected two species of mRNAs with the size of 5.2 and 2.7 Kb in CHO cells. However, the steady-state level of the two mRNAs was reduced 26-fold in the X-ray-sensitive mutants, xrs-5 and xrs-6. Several experiments were performed to study the mechanism of underexpression. The stability of B52 mRNA was the same in the K1 and xrs mutant cells. No detectable gross structural and copy number changes of the B52 gene were observed in the two xrs mutants. The LTR region of the B52 cDNA was able to initiate transcription of the reporter gene CAT in CHO cells. Interestingly, the promoter/enhancer activity of B52LTR was 5-10 fold lower when assayed in the xrs-5 mutant cells. Various controls indicated that the xrs-5 mutant had the same capacity to be transfected. Thus, the results strongly suggest that a cellular factor involved in the activation of the B52LTR is defective in the xrs-5 mutant. 11 genes were found underexpressed in the xrs mutants previously. The present finding provides an attractive model for studying the mechanism of underexpression of multiple genes in the xrs mutants. Characterization of the mutation will provide important information on the gene involved in rejoining DNA double-strand breaks.