The octamer-binding protein Oct3/4 was postulated to activate transcription through protein-protein interactions with hypothetical cellular coactivator(s). We have used a bacterially produced Oct3/4, as a protein-binding probe, to detect by far-Western assay the Oct3/4-associating proteins (OTAPs) from the embryonal carcinoma (EC) cells F9 and pachytene spermatocytes. Both common and cell-specific OTAPs were shown to interact directly with Oct3/4. Differentiation of the EC cells results in disappearance of most of OTAPs, supporting their coactivator nature. Several OTAPs detected in pachytene spermatocyte may represent germ cell-specific Oct3/4 coactivators.