The NK-type homeobox gene tinman and the MADS box gene D-mef2 encode transcription factors required for the development and differentiation of the Drosophila heart, and closely related genes regulate cardiogenesis in vertebrates. Genetic analyses indicate that tinman and D-mef2 act at early and late steps, respectively, in the cardiogenic lineage. However, it is unknown whether regulatory interactions exist between these developmental control genes. We show that D-mef2 expression in the developing Drosophila heart requires a novel upstream enhancer containing two Tinman binding sites, both of which are essential for enhancer function in cardiac muscle cells. Transcriptional activity of this cardiac enhancer is dependent on tinman function, and ectopic Tinman expression activates the enhancer outside the cardiac lineage. These results define the only known in vivo target for transcriptional activation by Tinman and demonstrate that D-mef2 lies directly downstream of tinman in the genetic cascade controlling heart formation in Drosophila.