The A element, a fourteen base pair sequence in the rabbit myosin heavy chain (HC) beta promoter (-276/-263), contains the M-CAT motif, a cis-acting element found in several muscle-specific genes. The A element is essential for muscle-specific transcription of the myosin HC beta gene. Recently, we have identified both muscle-specific and ubiquitous factors (A1 and A2 factors, respectively) that bind to the A element. Since the sequence of the A element is very similar to the GTIIC motif in the SV40 enhancer, we examined the relationship between A-element-binding factors and a GTIIC binding factor TEF-1, recently isolated from HeLa cells. The GTIIC motif was bound by the A1 and A2 factors in muscle nuclear extracts and competed with the A element for DNA-protein complex formation. Antibody against human TEF-1 'supershifted' the ubiquitous A2 factor-DNA complex, but did not alter the mobility of the muscle-specific A1 factor-DNA complex. We isolated a murine cDNA clone (mTEF-1) from a cardiac cDNA library. The clone is highly homologous to Hela cell TEF-1. The in vitro transcription/translation product of mTEF-1 cDNA bound to the A element, and the DNA binding property of mTEF-1 was identical to that of the A2 factor. Transfection of mTEF-1 cDNA into muscle and non-muscle cells confirmed that mTEF-1 corresponds to A2, but not to A1 factors. The mTEF-1 mRNA is expressed abundantly in skeletal and cardiac muscles, kidney and lung, but it is also expressed at lower levels in other tissues. These results suggest that the M-CAT binding factors consist of two different factors; the ubiquitous A2 is encoded by mTEF-1, but the muscle-specific A1 factor is distinct from mTEF-1.