Sex hormone-binding globulin (SHBG) transports sex steroids in the blood. In humans and rabbits, the gene encoding SHBG (shbg) is expressed primarily in the liver and testis, whereas the testis is the major site of shbg expression in rodents postnatally. Sequence analysis has revealed that rabbit shbg (rbshbg) spans 2.5 kb and comprises eight exons with consensus splice sites at all exon-intron junctions. The major transcription start site ofrbshbg is located 52 bp upstream from the translation initiation codon for the rabbit SHBG precursor. Unlike the situation in humans and rats, rbshbg transcripts contain no alternative exon 1 sequences in the liver or testis, and this suggests that the rbshbg 5'-flanking region plays an equally important role in controlling transcription of this gene in these tissues. Like the human and rat shbg promoter sequences, the rbshbg proximal promoter lacks a typical TATA box. It also contains several transcription factor-binding sites, but deoxyribonuclease I footprinting experiments indicated that the human and rabbit shbg proximal promoters interact quite differently with proteins extracted from rabbit liver nuclei. However, the predominant footprint on the rbshbg promoter is conserved at the same position within the human shbg (hshbg) promoter and includes consensus binding sites for the transcription factor nuclear factor- 1. Transient transfection studies of the rbshbg 5'-flanking sequence (893 bp) revealed regions that actively enhance and repress its activity in human hepatoblastoma and mouse Sertoli cells, but not in Chinese hamster ovary cells. Like the rat shbg proximal promoter, the rbshbg 5'-flanking sequence lacks a region that corresponds to a cis-element, designated footprinted region 4 in the hshbg proximal promoter. Furthermore, the hshbg promoter footprinted region 3 sequence is poorly conserved in rbshbg, and when mutated to resemble the corresponding human sequence it increased the transcriptional activity of the rbshbg promoter by 7-fold in hepatoblastoma cells. Thus, the rabbit and hshbg promoters appear to be controlled by a different set of transcriptional regulators. Further comparisons of their functional activities may shed light on species-specific differences in the spatial and temporal expression of this gene, the products of which play important roles in regulating sex steroid access to target cells.