Using extracts of AtT-20 cell nuclei, protein binding sites on the POMC gene 5'-flanking region were examined with an exonuclease protection approach. One such binding site, located from -119 to -106 bp upstream from the mouse POMC gene transcription initiation site, which exhibited a close homology to the activator protein-2 (AP-2) site [1]. A double-stranded oligonucleotide containing this site was subsequently used in gel shift assays to demonstrate AP-2 consensus sequence binding activity in extracts of AtT-20 cell nuclei. Gel shift competition experiments using both homologous and heterologous competitor DNA sequences revealed that the AP-2 like factor(s) exhibited specific binding to the mouse AP-2 consensus sequence. Furthermore, AP-2 factor binding was also modulated by a CTF/NF1-like factor. Pretreatment of AtT-20 cell nuclear extracts with alkaline phosphatase prior to inclusion in gel shift assays led to a reduction in the intensities of AP-2 factor-specific bands, indicating a potential involvement of protein phosphorylation in AP-2 factor binding in AtT-20 cells.