Human chorionic somatomammotropin (hCS) gene expression in the placenta is controlled by an enhancer (CSEn) containing SV40-related GT-IIC and SphI/SphII enhansons. These enhancers are controlled by members of the transcription enhancer factor-1 (TEF-1) family. Recently TEF-5, whose mRNA is abundant in placenta, was shown to bind cooperatively to a unique, tandemly repeated element in CSEn2, suggesting that TEF-5 regulates CSEn activity. However, expression of TEF-5 using a cDNA lacking the 5'-untranslated region and containing a modified translation initiation site was not accompanied by CSEn activation. Using nested, degenerate PCR primers corresponding to conserved TEF domains, several novel TEF-1-related cDNAs have been cloned from a human placental cDNA library. The open reading frame of one 3033-bp clone was identical to TEF-5 and contained 300- and 1423-bp 5'- and 3'-untranslated regions, respectively. The in vitro generated approximately 53-kDa TEF-5 polypeptide binds specifically to GT-IIC and SphI/SphII oligonucleotides. Overexpression of TEF-5 in BeWo cells using the intact 3033-bp cDNA transactivates the hCS and SV40 enhancers and artificial enhancers comprised of tandemly repeated GT-IIC enhansons, but not OCT enhansons. The data demonstrate that TEF-5 is a transactivator that is likely involved in the transactivation of CSEn enhancer function. Further, the data suggest that elements within the untranslated regions, initiation site, or both control TEF-5 expression in ways that influence its transactivation function.