The myocyte enhancer binding factor 2 (MEF2) family of transcription factors plays an important role in the regulation of gene expression in multiple muscle cell types. Four mef2 genes (A, B, C, and D) have been identified in vertebrates. They share the conserved N-terminal MCM1-agamous-deficiens-serum response factor and MEF2 domains that determine DNA binding and dimerization functions. In this study, we have identified human MEF2A domains that control its nuclear localization and transcriptional activation function. Studies of subcellular localization of various truncated MEF2A proteins expressed in HeLa cells demonstrated that MEF2A contains a nuclear localization signal located at its C terminus within the peptide encompassing amino acids (aa) 472-507. Examination of MEF2A mutants with sequential C-terminal deletions indicates that the region encompassing aa 321-472 is essential for transcriptional activity. Analysis of the transcriptional activity of fusion proteins consisting of different domains of MEF2A fused to the DNA binding domain of yeast transcription factor GAL4 revealed a major transcriptional activation domain located between aa 274 and 373. Further studies demonstrated that this domain contains positive and negative regulatory subdomains that cooperate with one another to regulate the transcriptional activity of MEF2A in a cell-type discriminatory manner.