We have examined the functional properties of a putative regulatory sequence, CCAAAAGTGG, (element A) in chicken cardiac myosin light chain 2 (MLC2) gene promoter by deletion/substitution mutagenesis and transcriptional analysis of RNA by S1 nuclease mapping. The results indicate that the element A sequence, which resembles the evolutionarily conserved A/T-rich CArG box, plays a role in defining the transcription initiation site in MLC2 gene. This is accomplished via repression of a potential transcription initiation at site -40 and promoting the initiation at +1. One of the two other dA-dT-rich sequences (element C), located proximal to initiation site (+1), serves as the basal promoter while the distal A/T rich element B participates in tissue specific transcription of the gene.