Expression of the gene encoding cytochrome P450 17alpha-hydroxylase, CYP17, is necessary for adrenal and gonadal steroidogenesis in most species. However, some animals, such as the pig, express CYP17 in the trophectoderm of the preattachment blastocyst, an event associated with estrogen synthesis and the establishment of pregnancy. How trophoblastic expression of CYP17 is regulated in the porcine blastocyst remains unknown and forms the basis of the following studies. The porcine CYP17 gene, including the complete coding and several kilobases of 5'-flanking regions, was cloned and sequenced. Blastocysts were examined by Northern analysis to verify the level of CYP17 transcript, and tissue-specific expression in the trophectoderm was confirmed by in situ hybridization. Primer extension, S1 nuclease protection, and 5'-rapid amplification of cDNA ends confirmed a common proximal transcription start site in adrenals and gonads (-48 bp) but identified a unique distal start site used in porcine trophectoderm (-182 bp). Additionally, reporter analysis of the CYP17 regulatory region demonstrated that constructs (-27 to -718 bp) were unresponsive to forskolin when expressed in porcine trophoblast cells, suggesting that trophoblast may not be able to respond to cAMP induction of this gene. The identification of this distal, previously undescribed, transcriptional start site suggests that unique mechanisms control the expression of CYP17 in porcine trophectoderm and possibly other genes important in implantation and early placental development.