The proximal promoter (-383/+16) of the ovine placental lactogen (oPL) gene provides trophoblast-specific expression in vitro. Footprint 6 (FP6; -319/-349) lies within this region, and transfection of two-base pair mutations across FP6 into BeWo cells identified potential binding sites for CCAAT-enhancer binding protein (CEBP) and specificity proteins (Sp). Transfection of CEBP dominant negative or over-expression constructs did not impact transactivation of the proximal promoter. However, Sp1 and Sp3 over-expression constructs increased (p<or=0.05) transactivation. Additionally, Sp1 and Sp3 short-hairpin RNA constructs reduced (p<or=0.01) transactivation of the proximal promoter. In EMSA supershift assays, Sp1 and Sp3 antibodies were able to inhibit migration of the complexes formed with nuclear extracts from BeWo cells and ovine chorionic binucleate cells (oBNC). Furthermore, Southwestern analysis of oBNC nuclear extracts identified a nuclear protein corresponding with Sp3, identified by Western analysis. In conclusion, these results indicate that Sp1 and Sp3 are capable of interacting with FP6 of the oPL gene proximal promoter and function to enhance its transactivation.