The factors that regulate the developmental expression of the rodent prolactin gene family in placenta remain poorly defined. We previously identified an enhancer element in the 5' flanking region of one family member, rat placental lactogen II (rPLII), which could target reporter gene expression to the placenta in transgenic mice; this enhancer functioned in the Rcho rat trophoblast cell line but not in the rat pituitary GC cell line. In further experiments to identify the factors that bind this element, we have selectively enriched for DNA binding proteins in nuclear extract from Rcho cells using magnetic beads coupled to a 43 bp enhancer oligonucleotide. Tryptic peptides of bound proteins were analyzed by high performance liquid chromatography coupled off-line to matrix assisted laser desorption ionization time of flight mass spectrometry. Several peptides of AP2, a key trophoblast cell-specific transcription factor, were identified. Gel mobility shift assays using AP2-specific antiserum, and mutant enhancer oligonucleotides demonstrated binding specifically to the FP2 DNAse I protected region of the element, identifying an atypical binding site for this factor. In co-transfection assays in rat pituitary GC cells, AP2 transactivated the enhancer via this region. Chromatin immunoprecipitation assays confirmed AP2 occupancy of the enhancer region in situ in the nuclei of Rcho giant cells. These data support a role for AP2 in the placental giant cell-specific expression of the rPLII gene and provide the first direct evidence for the involvement of a placental-specific transcription factor in the regulation of a member of this gene family.