We previously identified three separate cDNAs (KS6, KS32 and KS43) for the human reduced folate carrier (RFC) with unique 5' untranslated regions (5' UTRs) [Wong, Proefke, Bhushan and Matherly (1995) J. Biol. Chem. 270, 17468-17475]. Multiple RFC transcripts were confirmed in CCRF-CEM cells and transport-up-regulated K562.4CF cells by 5' rapid amplification of cDNA ends (5' RACE) and/or primer extension analysis. Two groups of 5' RACE clones were identified, one containing a variable length sequence identical with the KS43 cDNA 5' UTR, and another consisting of variants of the KS32 5' UTR, apparently generated by alternative splicing. The 5' UTR for the KS6 cDNA was not detected. A single band was detected on Southern blots of CCRF-CEM genomic DNA probed with a 326 bp genomic fragment common to all three cDNA species. The unique 5' UTRs for the KS43 and KS32 transcripts were localized to separate non-coding exons (exons 1 and 2 respectively), upstream from a large (approx. 3.42 kb) intron; the KS6 5'UTR also mapped to exon 1. Exons 1 and 2 were contiguous with 996 and 342 bp GC-rich 5' flanking regions (designated Pro43 and Pro32 respectively) that contained multiple SP1 and AP2 but no TATA or CAAT boxes. Both Pro43 and Pro32 exhibited strong promoter activities when cloned in front of a luciferase reporter gene and transfected into HT1080 and K562 cells. By an analysis of promoter deletion mutants we identified two 89 bp tandem repeats that seemed to increase Pro32 activity, and a 240 bp distal sequence that repressed Pro43 activity. Taken together, our results show that multiple human RFC transcripts are encoded by a single gene locus and that the heterogeneous 5' UTRs result from multiple transcriptional starts and variable splicing of alternative non-coding exons transcribed from separate promoters.