Transcriptional control of the cardiac/slow skeletal alkali myosin light-chain (MLC1c/1s) gene is mediated, in part, by two highly conserved AT-rich cis-acting elements present in the immediate 5' flanking region. These elements cooperate to form an enhancer that can impart tissue specificity to heterologous promoters that are themselves not tissue specific in their pattern of expression. In the chicken, one of these elements matches the binding site for myocyte-specific enhancer-binding factor 2, while the other is a cis-acting element present in the transcriptional control regions of all striated alkali MLC genes (except MLC3f) and is referred to as the MLC box. The central decanucleotide core region of the MLC box closely resembles the CArG (CC[A/T]6GG) box of the serum response element, and the binding of muscle nuclear protein complexes to this element can be competed for with a synthetic serum response element. On the basis of their competition profiles and requirements for nonspecific competitor, two nuclear protein complexes, which compete for binding to the CArG-like region of the MLC box, have been identified. One of the complexes binds to a mutation of the CArG-like region that inactivates transcription of a linked reporter gene, while binding of the other complex is inhibited by this mutation. This latter complex reacts with an antibody to serum response factor (SRF) and exhibits the same binding characteristics as purified SRF. These results demonstrate that transcriptional control of the chicken MLC1c/1s gene resides in an upstream enhancer that is composed of two separate AT-rich elements, both of which are required to drive expression of a linked reporter gene. The binding of a nuclear protein complex containing SRF to one of these elements, the MLC box, is required for gene activation and apparently inhibited by other nuclear factors whose binding overlaps that of the SRF complex.