The human reduced folate carrier (hRFC) gene has a complex regulation involving 6 alternatively spliced non-coding exons and promoters (A1/A2, A, B, C, D, and E). The hRFC-A1/A2 promoter is unique in that it transcribes a novel transcript with an in-frame AUG in non-coding exon A1/A2 that encodes a modified hRFC protein with altered transport function. In this report, we characterize the hRFC-A1/A2 promoter in HepG2 human hepatoma cells. By transfecting HepG2 cells with 5' and 3' deletion constructs, a transcriptionally important 270 bp region was identified. Gel shift assays identified transcription factor binding to three E-box elements and one GATA site within this region. These elements were verified by transfections of mutant constructs into HepG2 cells. Cotransfections in Drosophila Mel-2 cells confirmed promoter activation by USF1 and GATA1. A physical association between USF1 and GATA1 was demonstrated by their co-immunoprecipitation. By real time PCR analysis of transfected HepG2 cells, USF1 and GATA1 increased endogenous hRFC-A1/A2 transcripts. Altogether, our results demonstrate a transcriptionally important region in the hRFC-A1/A2 promoter including E-box and GATA elements, and a transactivation by USF1 and GATA1 proteins. Our results further establish the complexity of hRFC regulation, as a means of ensuring adequate folate cofactor transport for cell proliferation.