The primary structures of the human KB cell (FR-KB1) folate receptor (FR) and of a human placental (FR-P2) FR, proteins important in cellular accumulation of folates, have been deduced from cDNA sequences. Herein, we report a novel human FR cDNA (FR-P3) isolated from a placental library and the chromosomal organization of the human FR-P3 gene. Compared to the FR-P2 cDNA, the composite 1084 base-pair (bp) FR-P3 cDNA is homologous, but contains a unique 5' terminus and sequence differences within the open reading frame (ORF) and at the exon I-II junction. Polymerase chain reaction and RNase protection assays demonstrate that the FR-P3 cDNA represents the major transcript, and suggest that the FR-P2 cDNA is encoded by an independent FR gene. The nucleotide sequences of two non-overlapping human genomic clones contain the FR-P3 gene, which spans 5148 bp, is composed of five exons, and is polymorphic relative to 5' restriction sites. The transcript size (1084 bp) predicted from structural analysis of the FR-P3 gene correlates with the size (1100 bp) determined by Northern blots. Based on RNase protection assays, both FR-P3 and FR-KB1 transcripts are expressed in human fetal and adult tissues, and the abundance of each transcript varies among the tissues studied. These results indicate that the FR transcripts are products of independent, conserved genes; that neither FR gene is preferentially expressed during fetal development; and that specific FR transcripts are differentially expressed in human tissues, suggesting that transcription of each FR gene is regulated independently. The isolation of the FR-P3 gene will permit functional analysis of the cis and trans regulatory elements of the FR-P3 gene and the mechanisms involved in tissue-specific FR gene expression.