SHBG is a homodimeric plasma glycoprotein. It functions as a carrier for sex steroids in blood and regulates their access to target cells. In human and rabbit, SHBG is a single-copy gene comprised of eight exons and is expressed primarily in the liver and testis. In the present study, the ontogeny of rabbit SHBG (rbSHBG) gene expression was examined in both fetus and mothers. Trace amounts of rbSHBG mRNA were detected in fetal liver from d 11 to d 29 gestation. These levels increased dramatically at d 30 and remained high until parturition (d 33). In contrast, high levels of rbSHBG mRNA were detected in the maternal liver early during pregnancy, with maximal levels being attained by d 22 and declining markedly thereafter. A rbSHBG transcript lacking the exon 4 sequences was consistently expressed along with the rbSHBG mRNA. When expressed as a glutathione-S-transferase-fusion protein, this alternatively spliced rbSHBG transcript resulted in a product with almost no steroid binding activity, unlike the full-length rbSHBG-glutathione-S-transferase fusion protein, which bound 5alpha-dihydrotestosterone. Antibody specific to the novel rbSHBG isoform lacking the exon 4-encoding domain was raised, and a single immunoreactive protein of 33-35 kDa was detected by Western blot analysis in both fetal and maternal liver, and this indicates that the rbSHBG transcripts lacking exon 4 sequences are translated in vivo. An RT-PCR analysis further revealed that this alternatively spliced SHBG transcript is present in human HepG2 cells as well as human and mouse testes, indicating that exon 4 splicing in SHBG transcription is conserved among mammalian species. To our knowledge, this is the first report of the identification of a SHBG exon 4 splice variant that is translated. Because the SHBG isoform it encodes lacks appreciable steroid-binding activity, it may function beyond that of the widely accepted role of SHBG as a steroid-transport protein.