A novel mechanism of regulation of cardiac alpha and beta myosin heavy chain gene by naturally occurring antisense transcription was elucidated via pre-mRNA analysis. Herein, we report the expression of an antisense beta myosin heavy chain RNA in the normal rodent myocardium. The pattern of expression of the antisense betaMHC RNA (beta RNA) under altered thyroid state and in diabetes directly correlates with that of the alpha pre-mRNA/mRNA, whereas it negatively correlates with the beta mRNA expression. Rapid amplification of the 5' end shows that this antisense transcript originates 2 kb downstream of the beta gene, and it is transcribed across the entire beta gene from the opposite strand. Our results demonstrate that the beta-alpha myosin heavy chain intergenic DNA possesses a bidirectional transcriptional activity, one direction transcribing the alpha gene, and the opposite direction transcribing the antisense beta RNA. This process turns on the alpha expression, and it simultaneously turns off that of the beta and thus coordinates alpha and beta expression in an opposite fashion. Comparative analyses of the intergenic DNA sequence across five mammalian species revealed a conserved region that is proposed to be a common regulatory region for the alpha and antisense beta promoter. This finding unravels the mechanism of cardiac alpha-beta gene switching and implicates the role of cardiac myosin gene organization with their function.