Immunohistochemical analysis of myosin heavy chain (MHC) isoform expression in perinatal and adult rat diaphragm muscles was performed with antibodies which permitted the identification of all known MHC isoforms found in typical rat muscles. Isoform switching, leading to the emergence of the adult phenotype, was more complex than had been previously described. As many as four isoforms could be coexpressed in a single myofiber. Elimination of developmental isoforms did not usually result in the myofiber immediately achieving its adult phenotype. Activation of genes for specific adult isoforms might be delayed to puberty. For example, two of the three fast MHCs, MHC2x and MHC2A appeared perinatally, while MHC2B did not appear until 30 days postnatal. By Day 60 this isoform was present in ∼27% of the myofibers, but in most myofibers expression of this isoform was transient (i.e., at Day ≥ 115, <4% of the myofibers expressed MHC2B). Fibers which contained MHCβ/slow during the late fetal and early neonatal period coexpressed MHCemb. A marked increase in the frequency of fibers containing MHCβ/slow occurred between 4 and 21 days postnatal. These slow fibers arose from a population of myofibers which expressed MHCemb and MHCneo during their development, and they accounted for the majority of slow fibers found in the adult diaphragm. The adult myosin phenotype of the diaphragm myofibers (as determined with immunocytochemistry, and 5% SDS-PAGE) was not achieved until the rat was ≥115 days old.