Analysis of mRNA by Northern blot and reverse transcription-polymerase chain reaction demonstrated the expression of sense and considerable amounts of naturally occurring antisense mRNA for -myosin heavy chain (MHC) and ␣-MHC in the neonatal rat heart: antisense MHC mRNA expression of ␣-MHC and -MHC was approximately half of the corresponding sense MHC mRNA expression. Using a computational approach, we could identify a reverse Pol II promoter in the -MHC gene. Both sense and antisense MHC mRNA demonstrated similar sizes of approximately 6,000 bp in the Northern blot. Alpha-MHC antisense mRNA consisted of approximately 3,700 bp of complementary exon sequences and -MHC consisted of approximately 2,700 bp, suggesting a higher probability of ␣-MHC mRNA dimerization. Hence, sense mRNA transcripts and protein of ␣-MHC should exist at different relative levels in the neonatal state. In fact, the relative proportion of ␣-MHC was 52.0 Ϯ 2.6% on the sense mRNA but only 36.3 Ϯ 1.8% on the protein level. Because of its high abundance in the heart, we suggest that in the neonatal heart naturally occurring antisense mRNA may play a role in the regulation of MHC expression and, therefore, in the control of the energetical and contractile behaviour of the heart.