To study hormonal regulation of rat androgen-binding protein (ABP) we have cloned and sequenced the gene. A 5.3-kbp genomic DNA fragment was found to contain the entire coding region of the gene, which consists of 8 exons. The major site of transcription initiation in the testis was localized by primer extension and is located 36 bases upstream from the site of translation initiation. The gene does not contain a "TATA box" immediately upstream from the major start site. The sequence TACCTA occurs at residue -23, which is a functional TATA-like element in the SV40 major late gene. A sequence related to the cAMP response element is at residue -126 bp. Southern blot analysis of rat genomic DNA indicated a single gene for ABP in the rat. The existence of one gene supports the idea that sex steroid-binding protein (SBP) produced by fetal rat liver is coded by the same gene. The possibility that an alternate promoter region is active in the fetal liver is discussed.