Human TR2 orphan receptor, isolated from the testis and prostate, is a member of the steroid/thyroid hormone receptor superfamily. With the screening of a human genomic library and the combination of primer walking and PCR sequencing, we found that the entire TR2 orphan receptor gene coding region and 5'-untranslated region feature 13 introns and 14 exons, and that the consensus splice sequences (GT-AG) are present in all intron-exon boundaries. Within the region that codes for the DNA binding domain, TR2 orphan receptor gene has a distinct intron-exon junction. Whereas all other known steroid receptors have one splice site that separates their first and second zinc fingers in the DNA binding domain, TR2 orphan receptor has a rare splice site located in the middle of its first zinc finger. The identification of specific junction sequences for potential alternative splicing sites helps to explain the existence of multiple forms of TR2 orphan receptor cDNA (TR2-5, 7, 9, 11). The S1 nuclease protection assay for TR2 message revealed that there are multiple transcription initiations, and that the major cap site surrounded by an initiator-like sequence is located at the 104th nucleotide upstream from the translation start codon. Sequence analysis of a 2.7-kb DNA fragment upstream of the TR2 orphan receptor translation start codon unveiled several potential cis-acting elements, such as AP-1, HNF-5, GATA1 binding sites, and GC boxes. Using fluorescence in situ hybridization combined with a high-resolution G-banding technique, we found that the TR2 orphan receptor gene was mapped to human chromosome 12 at band q22, whereas the structurally closely related TR4 orphan receptor gene was mapped to human chromosome 3 at band q24.3.