The structural organization of the murine RFC-1 gene encoding a folate transporter has been determined. The entire nucleotide sequence of the L1210 cell RFC-1 cDNA, the 3'- and 5'-untranslated regions and the coding sequence were found to be distributed in eight exons, including six primary exons and alternates to exon 1 and exon 5, spanning 10.4 kb. Splice variants were identified in an L1210 cell cDNA library. The most common incorporates exons 1 through 6, encoding a 58-kDa polypeptide. The two least common incorporate exons 1 and 2, a truncated version of exon 3 and exons 4 through 6; or exons 1 through 4, an alternate to exon 5, and exon 6, encoding polypeptides of 53.6 and 43.4 kDa, respectively. A fourth variant reported earlier (GenBank/EMBL accession No. L36539) by others incorporates what we have found to be an alternate of exon 1 and exons 2 through 6. A relatively GC-rich region of the genome just 5' of exon 1 as well as exon 1a appears to be distinctly promoter-like and encodes a number of putative cis-acting elements. The findings pertaining to alternates of exon 1 suggest that the transcription of RFC-1 variants results from two different promoters.