To investigate the regulational interaction of hepatocyte nuclear factor-1α (HNF-1α) and insulin promoter factor 1 (IPF1) on insulin gene expression, either or both of the expression vectors carrying each transcription factor were transiently transfected into HeLa cells, RINm5F cells and MIN6 cells together with the luciferase reporter construct driven by a human preproinsulin gene promoter (−1998 to +237) designated as pINS-1998/luc. IPF1-transfection into HeLa cells strongly stimulated the luciferase activity to 725 fold that of the basal level. In contrast, HNF-1α-transfection resulted in only a 6.7 fold increase. In co-transfection experiments, increasing the amount of HNF-1α resulted in an 84.5% and 74.4% decrease in IPF1-stimulated luciferase activity in HeLa and RINm5F cells, respectively. Deletion constructs designated as pINS-248/luc, pINS-213/luc and pINS-185/luc were transfected into RINm5F cells to determine the role of the A3 element and its 5′ flanking sequence in the inhibitory effect of HNF-1α. The results showed that the inhibiting effects of HNF-1α with pINS-213/luc and pINS-185/luc were significantly smaller than those with both pINS-1998/luc and pINS-248/luc. Transfection into MIN6 cells with pINS-1998/luc in the absence of IPF1 resulted in constitutional transactivation of the insulin gene, and this transactivation was abolished by the co-transfection with HNF-1α. The present data indicate that IPF1 rather than HNF-1α predominantly transactivates the insulin gene, and that HNF-1α inhibits IPF1-dependent insulin gene transactivation mediated through the 5′ flanking sequence of the A3 element. It is suggested that HNF-1α may be involved in insulin gene expression as a negative regulator.