The human reduced folate carrier (hRFC) is ubiquitously but differentially expressed in human tissues and its levels are regulated by up to six alternatively spliced non-coding regions (designated A1/A2, A, B, C, D, and E) and by at least four promoters. By transient transfections of HepG2 human hepatoma cells with 5' and 3' deletion constructs spanning 2883 bp of upstream sequence, a transcriptionally important region was localized to within 177 bp flanking the transcriptional start sites for exon C. By gel shift and chromatin immunoprecipitation assays, Sp1 and C/EBP beta transcription factors were found to bind consensus elements (GC-box, CCAAT-box) within this region. The functional importance of these elements was confirmed by transient tranfections of HepG2 cells with hRFC-C reporter constructs in which these elements were mutated, and by co-transfections of Drosophila SL-2 cells with wild-type hRFC-C promoter and expression constructs for Sp1 and C/EBP beta. Whereas both Sp1 and C/EBP beta transactivated hRFC-C promoter activity, C/EBP alpha and gamma were transcriptionally inert. Sp1 combined with C/EBP beta resulted in a synergistic transactivation. In HepG2 cells, transfections with Sp1 and C/EBP beta both increased endogenous levels of hRFC-C transcripts. By 3' deletion analysis, a repressor sequence was localized to within 71 bp flanking the minimal promoter. On gel shifts, a novel transcriptional repressor was localized to within 30 bp. Collectively, these results identify transcriptionally important regions in the hRFC-C minimal promoter that include a GC-box and CCAAT-box, and suggest that cooperative interactions between Sp1 and C/EBP beta are essential for hRFC-C transactivation. Another possible factor in the tissue-specific regulation of the hRFC-C region involves the downstream repressor flanking the minimal promoter.