During early postnatal development in rat diaphragm muscle (DIAm), significant fiber growth and transitions in myosin heavy chain (MHC) isoform expression occur. Similar to other skeletal muscles, DIAm fibers are multinucleated, and each myonucleus regulates the gene products within a finite volume - myonuclear domain (MND). We hypothesized that postnatal changes in fiber cross-sectional area (CSA) are associated with increased number of myonuclei so that the MND size is maintained. The DIAm was removed at postnatal days 14 (P-14) and 28 (P-28). MHC isoform expression was determined by SDS-PAGE. Fiber CSA, myonuclear number and MND size were measured using confocal microscopy. By P-14, significant coexpression of MHC isoforms was present with no fiber displaying singular expression of MHC_Neo. By P-28, singular expression was predominant. MND size was not different across fiber types at P-14. Significant fiber growth was evident by P-28 at all fiber types (fiber CSA increased by 61%, 93% and 147% at fibers expressing MHC_Slow, MHC_2A and MHC_2X, respectively). The number of myonuclei per unit of fiber length was similar across fibers at P-14, but greater at fibers expressing MHC_2X at P-28. The total number of myonuclei per fiber also increased between P-14 and P-28 at all fiber types. Accordingly, MND size increased significantly by P-28 at all fiber types, and became larger at fibers expressing MHC_2X compared to fibers expressing MHC_Slow or MHC_2A. These results suggest that MND size is not maintained during the considerable fiber growth associated with postnatal development of the DIAm.