Transcriptional Enhancer Factor-1 (TEF-1) is a transcription factor required for cardiac muscle gene activation. Since ablation of TEF-1 does not abolish cardiac gene expression, we sought to identify a human gene related to TEF-1 (RTEF-1) that might also participate in cardiac gene regulation. A human heart cDNA library was screened to obtain a full-length RTEF-1 cDNA. Fluorescence in situ hybridization assigned the RTEF-1 gene to chromosome 12p13.2-p13.3. In contrast, PCR screening of human/rodent cell hybrid panels identified TEF-1 on chromosome 11p15.2, between D11S1315 and D11S1334, extending a region of known synteny between human chromosomes 11 and 12 and arguing for an ancient divergence between these two closely related genes. Northern blot analysis revealed a striking similarity in the tissue distribution of RTEF-1 and TEF-1 mRNAs; skeletal muscle showed the highest abundance of both mRNAs, with lower levels detected in pancreas, placenta, and heart. Phylogenetic analysis of all known TEF-1-related proteins identified human RTEF-1 as one of four vertebrate members of this multigene family and further suggests that these genes diverged in the earliest metazoan ancestors.