The pivotal role of apolipoprotein AI (Apo AI) in mediating reverse cholesterol transport has lead us to the study of transcription factors that influence the expression of this gene. Previous studies show that rat HNF-4 enhances the activity of a cis-acting site C in the rat Apo AI promoter. Since sites C and A share 80% homology, we have examined whether HNF-4 binds to and modulates the transcriptional activity of the A-motif. Results show that HNF-4 binds to site A. The transcriptional activity of site A in a human hepatoma cell line, HuH-7, increases 2-2.5-fold in the presence of antisense HNF-4, but the sense construct has no effect on the activity of the reporter template. The lack of an effect of HNF-4 on site A activity may be due to high endogenous levels of the factor in HuH-7 cells. However, in BHK cells HNF-4 clearly inhibits the transcriptional activity of site A. Together these findings suggest that in contrast to the enhancing effects of HNF-4 on site C, the same factor inhibits site A activity. Since hepatocytes normally contain the T3 receptor and this nuclear factor increases site A action, cotransfection of T3 receptor along with antisense HNF-4 further augments the activity of p5'A.CAT. In summary, rat HNF-4 binds to site A from rat Apo AI DNA, and this factor suppresses site A activity. HNF-4 interferes with the enhancer role of the T3 receptor and thus contributes negatively to the net expression of the Apo AI gene.