We isolated a cDNA clone, Elk-3, that encodes a novel Ets transcription factor from 16-day mouse embryos. The deduced amino acid sequence of the protein was homologous to human ELK-1 and SAP-1. This protein, ELK-1, and SAP-1 shared some unique structural properties such as an Ets DNA-binding site in the amino-terminal region, a serum response factor interacting domain and phosphorylation sites of serine or threonine residues in the carboxy-terminal region. Northern blotting weakly revealed that two transcripts of 4 and 2.1 kb are expressed in the adult ovary and lung and a 2.1-kb transcript predominated in 8- to 14-day embryos. We assayed the transcriptional activities of Elk-3 protein on the cytokeratin EndoA enhancer containing Ets binding sites in endodermal cells. Elk-3 protein strongly repressed enhancer activity but did not affect the activity of the basal promoter in the absence of the enhancer. Furthermore, Elk-3 can suppress the activity of Ets-2 as the transcriptional activator on the EndoA enhancer. These data suggested that the Elk-3 gene product plays a role in transcriptional regulation during embryogenesis.