The role of estrogens in the development and physiology of the male reproductive tract remains provocative, with a growing body of evidence suggesting that estrogens are able to influence normal testis development and physiology, through their classical receptors, estrogen receptor (ER)-alpha and ER-beta. We describe the identification and characterization of a new promoter that is involved in the expression of ER-alpha in the epididymis and in testis. This promoter lies on chromosome 6q25.1, approximately 16 kb upstream of the first coding exon of ER-alpha. Sequence analysis indicates that this promoter has a conventional TATA box and GC box but no upstream CAAT sequence. Alternative splicing results in at least two species of mRNA encoding ER-alpha being synthesized from this promoter. Transcription profiling of human tissues shows that, among those tested, this promoter is predominantly active only in testis and epididymal tissues. Transient transfection assays using this new promoter in a number of cell lines indicate that the region we have identified functions as a promoter and that tissue-specific regulation is likely to be dependent on inhibitory sequences greater than 1 kb upstream of the transcription start site.