Myogenesis is accompanied by the activation of two developmental myosin heavy chains (MyHCs), embryonic and perinatal, followed by a dramatic decrease in their expression during early postnatal life. The pathways that control the transcription of these genes have not been previously determined. In this study, we identified cis-acting elements and trans-acting factors that regulate the expression of these two developmental MyHCs in the mouse. Between 800 and 1000 bp of proximal promoter sequence is sufficient to drive muscle-specific expression in cell culture. Further, these same regions contain sequences that confer downregulation in postnatal life in vivo. For the embryonic MyHC gene, the region between -791 bp and -626 bp contains the majority of activating elements. In the proximal promoter regions of both genes, we identified two E-box elements that work in conjunction to activate transcription, but only the embryonic MyHC E-boxes bind a complex containing MyoD. In addition, our results reveal activation by calcineurin that is transduced only partially by its conventional downstream effectors, MEF2 and NFAT. Some common features are shared between the promoters of these two genes; however, the mechanisms of their regulation appear distinct.