The expression of the prolactin receptor is under the control of two putative tissue-specific (PI, gonads; PII, liver) and one common (PIII) promoters (Hu, Z. Z., Zhuang, L., and Dufau, M. L. (1996) J. Biol. Chem. 271, 10242-10246). The three promoter regions were co-localized to the rat chromosomal locus 2ql6, in the order 5'-PIII-PI-PII-3'. To investigate the mechanisms of gonad-specific utilization of PI, the promoter domain, regulatory cis-elements, and trans-factors were identified in gonadal cells. The promoter domain localized to the 152-base pair 5' of the transcriptional start site at -549 is highly active in gonadal cells but has minimal activity in hepatoma cells. It contains a steroidogenic factor 1 (SF-1) element (-668) that binds the SF-1 protein of nuclear extracts from gonadal cells and is essential for promoter activation. A CCAAT box (-623) contributes minimally to basal activity in the absence of the SF-1 element, and two adjacent TATA-like sequences act as inhibitory elements. Thus, PI belongs to a class of TATA-less/non-initiator gene promoters. These findings demonstrate an essential role for SF-1 in transcriptional activation of promoter I of the prolactin receptor gene, which may explain the tissue-specific expression of PI in the gonads but not in the liver and the mammary gland.