We describe the molecular cloning of murine (m) Transcriptional Enhancer Factor (TEF)-5 belonging to the TEF family of transcription factors. We show that mTEF-5 is specifically expressed in trophoblast giant cells and other extra-embryonic structures at early stages of development. At later stages, mTEF-5 is specifically expressed in the labyrinthine region of the placenta and in several embryonic tissues. We further show that the other mTEFs are differentially expressed in extraembryonic structures and in the mature placenta. Interestingly, human (h)TEF-5 is specifically expressed in the differentiated syncytiotrophoblast of the human term placenta and its expression is upregulated during the differentiation of cytotrophoblasts to syncytiotrophoblast in vitro, whereas that of hTEF-1 is down-regulated. Together with previous results describing hTEF-binding sites in the human placental lactogen-B gene enhancer, these novel observations support a role for hTEF-5 in the regulation of this gene. We further propose that the hTEF factors may play a more general role in placental gene regulation and development.