Different portions of the 5'-upstream region of the mouse proliferating cell-nuclear-antigen (PCNA) gene were combined with the bacterial chloramphenicol acetyltransferase (CAT) gene of a CAT vector. A transient expression assay of CAT activity in mouse neuroblastoma N18TG2 cells transfected with these recombinant plasmids and RNase protection analysis have revealed the existence of a negative regulatory region between nucleotides -1231 and -624 (+1 denotes the transcription initiation site). The CAT expression levels were gradually increased, depending on the extent of deletion from the 5'-terminus in this region, suggesting that the negative regulatory region consists of multiple elements with rather weak repressing activities. Significant sequence similarity was found between the negative regulatory region of the PCNA gene and those of the several reported genes. A 752-bp segment containing this negative regulatory region repressed the function of the PCNA gene promoter in an orientation-independent and position-independent manner. However, the negative regulatory region showed almost no repressing effect on the functions of the heterologous gene promoters such as the simian virus 40 enhancer promoter, the enhancer promoter in the Rous sarcoma virus long-terminal repeat and the mouse DNA polymerase beta gene promoter. These results suggest that the negative regulatory region of the mouse PCNA gene functions specifically to its own promoter. This unique property is discussed in comparison with that of the negative regulatory elements of the mouse DNA polymerase beta gene.