A culture model for cardiac hypertrophy, stimulation of neonatal rat cardiac myocytes by alpha(1)-adrenergic agonists, has been used extensively to identify transcription factors that activate genes during cardiac hypertrophy, such as skeletal alpha-actin, beta-myosin heavy chain (betaMHC), and B-natriuretic peptide. We used this culture model to further investigate transcription factors regulating the betaMHC promoter in cardiac myocytes under basal conditions and during hypertrophy. We found that the rat betaMHC promoter contains two other MCAT sites, in addition to the two MCATs reported previously. The four MCAT sites are conserved in some but not all of the mammalian betaMHC promoters examined, and all bind TEF-1 but with varying affinity. As assayed by transient transfection into cardiac myocytes, the four MCATs within 348 bp of the transcription start site are required for full activity of the rat betaMHC promoter in the absence and presence of the alpha(1)-adrenergic agonist phenylephrine (PE). We found that the betaMHC promoter also contains a binding site for the NFAT family of transcription factors, which are activated by calcineurin and are implicated in the hypertrophic process. Although this site bound NFAT3 in vitro and has been reported to be required for betaMHC promoter activity in slow skeletal muscle, mutation of the site had no effect on basal or on PE-induced activity of the promoter in cardiac myocytes. Our results show that full activity of minimal betaMHC promoters in the presence and absence of hypertrophic agents requires multiple MCAT sites but not NFAT-binding sites.