The transcriptional enhancer factor-1 (TEF-1) is a member of the TEA/ATTS domain family. TEF-1 binds to GT-IIC (GGAATG), SphI (AGTATG), SphII (AGCATG), and M-CAT (GGTATG) response elements and is involved in the transactivation of a variety of genes, including the SV40 large T antigen, mammalian muscle-specific genes, and human chorionic somatomammotropin genes. Also, TEF-1 acts as a transcriptional repressor in placental cells, possibly through interaction with the TATA binding protein (TBP), preventing TBP binding to the TATA box. Here we describe the cloning, tissue-specific expression pattern, and functional characterization of two novel TEF-1 isoforms, TEF-1beta and TEF-1gamma. These isoforms most likely arise from alternative splicing of mRNA transcribed from a single gene and involve substitutions and/or insertions in a region immediately following the DNA binding domain. TEF-1beta appears to be widely distributed like the prototypic TEF-1, designated TEF-1alpha, whereas TEF-1gamma exhibits a narrower tissue-specific expression pattern that includes pancreas, kidney, and skeletal and heart muscle. The relatively limited sequence alterations among these isoforms cause significant changes in their DNA binding and transcriptional activities. TEF-1beta and TEF-1gamma bind to GT-IIC sequences with higher affinity and repress hCS promoter more efficiently than TEF-1alpha. These results suggest that each TEF-1 isoform may play unique regulatory roles in various tissues.