Placental lactogen (PL) is thought to alter maternal metabolism to increase the pool of nutrients available for the fetus and to stimulate fetal nutrient uptake. The ovine (o) PL gene is expressed in chorionic binucleate cells (oBNC) and cis-elements located within the proximal promoter (-124 to +16 bp) are capable of trophoblast-specific expression in human (BeWo) and rat (Rcho-1) choriocarcinoma cells. Protein-DNA interactions were identified with oBNC nuclear extracts, and mutational analysis of these regions revealed a previously undefined cis-element from -102/-123 bp that enhances promoter activity in BeWo cells but not Rcho-1 cells. Characterization of this region identified the nucleotide sequence CCAGCA (-105/-110; o110) as the responsible cis-acting element. Southwestern analysis with this element identified a binding protein with an apparent M(r) of approximately 41,000. Expression screening of an ovine placental cDNA library identified six homologous cDNAs, which shared identity with human (97%) and mouse (95%) Pur alpha, a single-stranded DNA binding protein. The Pur alpha-o110 interaction was confirmed by electrophoretic mobility-supershift assays with oBNC and BeWo extracts but was absent with Rcho-1 extracts. Furthermore, overexpression of ovine Pur alpha enhanced transactivation of the oPL gene proximal promoter in both choriocarcinoma cell lines through this novel cis-element. This study identified a previously undefined cis-element, which interacts with Pur alpha to augment PL gene transcription.