The rat CYP1A1 negative regulatory element (NRE) contains AP-1 and Oct-1 motifs at -808 to -788 bp. The CYP1A1 sequence from -813 to -779 bp and an identical sequence bearing a point mutation in the octamer motif were synthesized. Gel mobility shift assays showed the formation of two complexes with the wild-type CYP1A1 sequence and nuclear extracts from H4IIE and HepG2 hepatoma cells and from rat liver. The formation of the major complex was significantly reduced with the mutant octamer-containing oligomer and was specifically competed by an Oct-1 oligodeoxyribonucleotide. The addition of Oct-1 antibody caused a supershift of the major complex. The presence of the wild-type sequence, but not the mutant octamer sequence, caused a 3-fold decrease in SV40 enhancerless promoter activity in transfected HepG2 cells. Co-transfection of an Oct-1 expression vector with rat CYP1A1 NRE octamer-containing, promoter/reporter gene constructs specifically further decreased promoter activity of the wild-type octamer-containing constructs in HepG2 cells. The results indicate that Oct-1 binds to the rat CYP1A1 promoter NRE and is a negative regulator of rat CYP1A1 expression.