A murine cardiac specific homeoboxgene, Nkx-2.5/CSX, a potential Drosophila tinman homologue, may have a fundamental role in cardiac myocyte differentiation. DNA binding targets for Nkx-2.5 were recently shown to represent novel homeodomain binding sequences, some of which resembled serum response elements (SREs); [Chen CY, Schwartz RJ (1995): J Biol Chem 270: 15628-15633]. In this study, Nkx-2.5 facilitated serum response factor (SRF) DNA-binding activity to the multiple SREs found on the cardiac alpha-actin promoter and together stimulated cardiac alpha-actin promoter dependent transcription in 10T1/2 fibroblasts. Analysis of cardiac alpha-actin promoter mutants demonstrated the importance of the multiple upstream SREs and an obligatory requirement for an intact proximal SRE1, for providing high levels of activity in the presence of Nkx-2.5 and SRF coexpression. Transfection assays with mutant SRF species indicated that the C-terminal activation domain and DNA-binding MADS box were necessary for transcriptional activity in the presence of Nkx-2.5. Expression of Nkx-2.5 mutants also demonstrated that the homeodomain alone was insufficient for directing promoter activity in the presence of SRF. The central role of SRF in regulating striated alpha-actin gene activity also was revealed by its embryonic expression restricted primarily to myocardium of the developing heart and the myotomal portion of somites. Thus the function of the cardiac actin promoter SREs appeared to provide binding sites for SRF and Nkx-2.5 to interact and elicit striated muscle specific transcription that was independent of the MyoD family.