Decreased reduced folate carrier (RFC) activity has been associated with MTX resistance in experimental models of transport-mediated MTX resistance, and has been attributed to changes in the expression of RFC1, the gene that encodes a protein with this activity. RNA transcripts of RFC1 may contain any one of four distinct 5' untranslated regions (UTRs). We cloned a human genomic DNA fragment upstream from the RFC1 translation start site and determined the nucleotide sequence of a 4.8kb region that contained the exons corresponding to each of the reported UTRs. To identify regulatory elements that may be involved in RFC1 transcription, we first developed a semi-quantitative RT-PCR assay using primers specific for each of the 5' UTRs to amplify RNA transcripts containing each of the RFC1 5' exons, and found evidence for differential transcription of RFC1 non-coding exons in tissues, during development, and in MTX-resistant, transport-deficient cells. We also found that RFC1 RNA levels are cell cycle regulated and peak at the G1 to S transition. Then, using a series of RFC1 promoter-reporter fusion constructs, we identified genomic sequences that may be involved in the regulation of expression of exons 1b and 1c of the RFC1 gene. These studies therefore identify regulatory regions of RFC1 promoters and potential models of regulation in which these regions may exert control.