Serum response factor (SRF) is a phosphoprotein that regulates skeletal and cardiac alpha-actin gene transcription. Myotonic dystrophy protein kinase (DMPK), a muscle- and neuron-restricted kinase, enhanced SRF-mediated promoter activity of the skeletal and cardiac alpha-actin genes in C2C12 myoblasts as well as in nonmyogenic cells. DMPK phosphorylated SRF in vitro in the alphaI coil of the DNA-binding domain in the MADS box, a highly conserved region required for DNA binding, dimerization, and co-activator interaction in COS and CV1 cells. Threonine 159 in the MADS box alphaI coil was a specific phosphorylation target in vitro as well as in vivo of both DMPK and protein kinase C-alpha. Substitution of threonine 159 with the nonphosphorylatable residue alanine markedly diminished activation of the cardiac alpha-actin promoter in the presence of kinase, while its substitution with aspartic acid, to introduce a negative charge and mimic phosphorylation, restored activation completely. Phosphorylation of the MADS box may constitute a novel mechanism for regulation of SRF-dependent actin gene transcription.