We have sought direct evidence for the in vivo expression of the human growth hormone-variant (hGH-V) gene by screening a placental cDNA library with a hGH-V-specific oligonucleotide. Nine independent hGH-V cDNA clones were isolated and analyzed, and three distinct species were detected. Five of these hGH-V cDNAs represent mRNAs spliced and processed in a pattern analogous to that of the highly homologous human growth hormone and human chorionic somatomammotropin gene transcripts. Each of the remaining four hGH-V cDNAs contains an additional segment of 253 nucleotides corresponding in position and sequence to the fourth intron of the hGH-V gene. In addition, one of the mRNAs in this second group uses an alternative downstream polyadenylation site. The alternatively spliced hGH-V mRNA, which we refer to as hGH-V2 mRNA, constitutes approximately 30% of the hGH-V transcripts both in the human term placenta and in a stable mouse fibroblast line expressing the transfected hGH-V gene. The placental expression of the hGH-V gene is specific to villous tissue. The hGH-V2 mRNA is predicted to encode a protein which substitutes the 65 carboxyl-terminal amino acids of hGH-V with a new 104-residue carboxyl terminus resulting in significant divergence in their relative physical properties. The alternative splicing of the hGH-V transcripts to hGH-V and hGH-V2 mRNAs expands the potential complexity of the hGH-V gene's role in normal placental function.