The PEA3 motif, first recognized in the polyomavirus enhancer, is an oncogene, serum growth factor, and phorbol ester-responsive element. An activity capable of binding to this sequence, termed PEA3 (polyomavirus enhancer activator 3), was identified in mouse 3T6 cell nuclear extracts. We have cloned cDNAs that encode PEA3 from a mouse FM3A cell cDNA library. A continuous open reading frame in the longest cDNA predicts a 555-amino-acid protein with a calculated molecular mass of 61 kD. Recombinant PEA3 binds to DNA with the same sequence specificity as that endogenous to FM3A cells and activates transcription through the PEA3 motif in HeLa cells. Deletion mapping of the protein revealed that the DNA-binding domain is located within a stretch of 102 amino acids near the carboxyl terminus. This region shares extensive sequence similarity with the ETS domain, a conserved protein sequence common to all ets gene family members. PEA3 is encoded by a 2.4-kb mRNA that is expressed to differing extents in fibroblastic and epithelial cell lines but not in hematopoietic cell lines. In the mouse, PEA3 expression is highly restricted; only the epididymis and the brain contain readily detectable amounts of its mRNA. Interestingly, the amount of PEA3 mRNA is down-regulated during retinoic acid-induced differentiation of mouse embryonic cell lines. These findings suggest that PEA3 plays a regulatory role during mouse embryogenesis.