Myosin is found in all eukaryotic cells and appears to be involved in diverse cellular motile processes such as cytokinesis, cell migration and cell adhesion. There are at least two genes for vertebrate nonmuscle myosin heavy chains (MHCs), MHC-A and B. The nonmuscle MHC-A gene is expressed abundantly in fibroblasts, epithelial cells and lymphoid cells, but less abundantly in neuronal cells and differentiated muscle cells. We have been studying the mechanisms responsible for the cell type-dependent transcription for the nonmuscle MHC-A gene. We have previously identified three clustered cis-regulatory elements (A, C and F) in intron 1, which modulates transcription in a cell type-dependent manner. One of these elements (element F) contains an E-box sequence. In this study, we undertook to identify and characterize trans-acting factors responsible for element F-mediated regulation. Yeast one-hybrid screening using element F allowed isolation of cDNAs encoding transcriptional factors TFEC, TFE3 and USF2, each of which contains basic helix-loop-helix and leucine zipper motifs. Furthermore, cDNA cloning by polymerase chain reaction yielded cDNAs for two TFEC isoforms, designated TFEC-1 and -s, which are most likely generated by alternative pre-mRNA splicing. In addition to these four factors, USF1, which is known to share the same DNA binding elements with USF2, was isolated for comparison. Electrophoretic mobility shift assays and cotransfection studies of the expression constructs with reporter gene constructs revealed that the above five factors have different binding activities for element F with different transactivation potencies. USF1 and USF2 demonstrate the highest binding activity to element F, yet show the lowest element F-dependent transactivation. TFE3 has a high transactivation potency, but the lowest binding activity. TFEC-l demonstrates a high binding activity with the highest transactivation potency, whereas TFEC-s has the same binding activity as TFEC-l with intermediate transactivation. TFE3 and TFEC (l or s) can form heterodimers and these heterodimers bind element F stronger than a homodimer of TFE3. These results suggest that TFEC-l is a major factor, together with TFE3, for activation of the nonmuscle MHC-A transcription via element F. We also found that the N-terminal activation domain exists only in TFEC-l, whereas the C-terminal activation domain is common to both the l and s isoforms. This study provides the first evidence of TFEC being an activator of transcription, with two separate activation domains.