The evolutionarily conserved order of the skeletal muscle myosin heavy chain (MHC) genes and their close tandem proximity on the same chromosome are intriguing and may be important for their coordinated regulation. We investigated type II MHC gene regulation in slow-type muscle fibers undergoing a slow to fast MHC transformation in response to inactivity, 7 days after spinal cord isolation (SI) in rats. We examined the transcriptional products of both the sense and antisense strands across the IIa-IIx-IIb MHC gene locus. A strand-specific reverse transcription (RT)-PCR approach was utilized to study the expression of the mRNA, the primary transcript (pre-mRNA), the antisense RNA overlapping the MHC genes, and both the intergenic sense and antisense RNAs. Results showed that the mRNA and pre-mRNA of each MHC had a similar response to SI, suggesting regulation of these genes at the transcriptional level. In addition, we detected previously unknown antisense strand transcription that produced natural antisense transcripts (NATs). RT-PCR mapping of the RNA products revealed that the antisense activity resulted in the formation of three major products: aII, xII, and bII NATs (antisense products of the IIa, IIx, and IIb genes, respectively). The aII NAT begins in the IIa-IIx intergenic region in close proximity to the IIx promoter, extends across the 27-kb IIa MHC gene, and continues to the IIa MHC gene promoter. The expression of the aII NAT was significantly up-regulated in muscles after SI, was negatively correlated with IIa MHC gene expression, and was positively correlated with IIx MHC gene expression. The exact role of the aII NAT is not clear; however, it is consistent with the inhibition of IIa MHC gene transcription. In conclusion, NATs may mediate cross-talk between adjacent genes, which may be essential to the coordinated regulation of the skeletal muscle MHC genes during dynamic phenotype shifts.Skeletal muscle is highly adaptable when subjected to altered loading and hormone states. Its size, metabolic makeup, and contractile properties can all be altered to optimize function (1). Variability in contractile properties is achieved mainly by diversification in the motor protein myosin heavy chain (MHC), 2 where different isoforms are encoded by distinct genes (1, 2). Of this family of eight MHC genes, six are tandemly linked and span ϳ420 kb in the rat on chromosome 10, with embryonic MHC situated at the most 5Ј end, sequentially followed by IIa, IIx, IIb, neonatal, and extraocular MHC.  (or type I) and ␣ MHCs are located tandemly on separate chromosomes (chromosome 14 in the rat); they span ϳ50 kb and are separated by 4.5 kb. Interestingly, the genomic order and orientation on the chromosomes of the MHC genes are conserved in all mammalian species, leading researchers to suspect that this organization might be an important feature in the strategy for the coordinated regulation of these genes (2-5).Types I, IIa, IIx, and IIb, in respective order of increasing ATPase activity, are the four predominately exp...