MHox is a mesoderm-specific homeodomain protein that binds an A/T-rich element that is essential for activity of the muscle creatine kinase (MCK) enhancer. The MHox binding site also binds the ubiquitous homeodomain protein Oct-1 as well as myocyte enhancer-binding factor-2 (MEF2), which belongs to the MADS superfamily of transactivators. To determine which of these proteins activates MCK transcription through the A/T element, we mutated this sequence such that it would selectively bind MHox, MEF2, or Oct-1 and tested the activities of the mutant enhancers in skeletal muscle cells. These mutant enhancers revealed that only MEF2 is able to activate the MCK enhancer through the A/T element. The convergence of homeodomain and MADS proteins on the A/T element in the MCK enhancer provides a mechanism through which a single DNA sequence can mediate positive and negative regulation of gene transcription and is reminiscent of the roles of these two classes of transcription factors in the control of other cell-specific genes.