Expression of the cardiac myosin heavy chain (MHC) genes is regulated developmentally and by numerous epigenetic factors. Here we report the identification of a cis-regulatory element and cognate nuclear binding protein(s) responsible for cAMP-induced expression of the rat cardiac alpha-MHC gene. By Northern blot analysis, we found that, in primary cultures of fetal rat heart myocytes, the elevation of intracellular levels of cAMP results in up-regulation of alpha-MHC and down-regulation of beta-MHC mRNA expression. This effect of cAMP was dependent upon the basal level of expression of both MHC transcripts and was sensitive to cycloheximide. In transient expression analysis employing a series of alpha-MHC/CAT constructs, we identified a 31-base pair fragment located in the immediate upstream region (-71 to -40), which confers both muscle-specific and cAMP-inducible expression of the gene. Within this 31-base pair fragment there are two regions, an AT-rich portion and a hybrid motif which contains overlapping sequences of E-box and M-CAT binding sites (GGCACGTGGAATG). By substitution mutation analysis, both elements were found important for the basal muscle-specific expression; however, the cAMP-inducible expression of the gene is conferred only by the E-box/M-CAT hybrid motif (EM element). Using mobility gel shift competition assay, immunoblotting, and UV-cross-linking analyses, we found that a protein binding to the EM element is indistinguishable from the transcription enhancer factor-1 (TEF-1) in terms of sequence recognition, molecular mass, and immunoreactivity. Methylation interference and point mutation analyses indicate that, besides M-CAT sequences, center CG dinucleotides of the E-box motif CACGTG are essential for protein binding to the EM element and for its functional activity. Furthermore, our data also show that, in addition to TEF-1, another HF-1a-related factor may be recognized by the alpha-MHC gene EM element. These results are first to demonstrate transcriptional activation of a sarcomeric gene by cAMP and support the role of TEF-1 and HF-1a-like factors in the regulation of alpha-MHC gene expression in cardiac myocytes.