During early postnatal development in rat diaphragm muscle (DIAm), significant transitions in myosin heavy chain (MHC) isoform expression occur which are associated with fiber growth and increased MHC protein. At present, there is no direct information regarding the transcriptional regulation of MHC isoform expression during postnatal DIAm development. We hypothesized postnatal changes in MHC isoform mRNA expression are followed by concomitant changes in MHC protein expression. The DIAm was removed at postnatal days 0, 14, 28 and 84 (adult). MHC mRNA expression was determined by real-time RT-PCR. MHC protein expression was determined by SDS-PAGE. There was a significant effect of postnatal age on MHC isoform mRNA and protein expression. At birth, the MHC_Neo isoform accounted for 28% of MHC mRNA and 54% of total MHC protein. By postnatal day 14, MHC_Neo mRNA and protein increased significantly and both decreased significantly by day 28, consistent with transcriptional control of the expression of this developmental isoform. By postnatal day 28, there were minimal changes in mRNA expression for MHC_Slow and MHC_2X, yet protein expression increased significantly. MHC_2A mRNA and protein expression did not change during this time. Thus, changes in MHC protein expression did not follow (or parallel) changes in MHC mRNA for the adult MHC isoforms. The present findings indicate that changes in MHC expression in the developing rat DIAm are not driven solely by changes in mRNA expression. Knowledge of isoform-specific MHC mRNA expression only yields predictive information on MHC protein expression for the MHC_Neo isoform.