The effects of 14 days of spaceflight on myonuclear number, fiber size, and myosin heavy chain (MHC) expression in isolated rat soleus muscle fiber segments were studied. Single soleus muscle fibers from rats flown on the Spacelab Life Sciences-2 14-day mission were compared with those from age-matched ground-based control rats by using confocal microscopy and gel electrophoresis. Spaceflight resulted in a significant reduction in the number of fibers expressing type I MHC and an increase in the number of fibers expressing type IIx or IIa MHC. Space-flight also resulted in an increase in the percentage of fibers coexpressing more than one MHC and in the reexpression of the neonatal isoform of MHC in some fibers. Fiber cross-sectional area was significantly reduced in pure type I MHC-expressing fibers and in fibers coexpressing type I+II MHC but not in fibers expressing one or more type II MHC in the flight rats. The number of myonuclei per millimeter was significantly reduced in type I MHC-expressing fibers from the flight rats but was not significantly different in type I+II and type II MHC-coexpressing fibers. Fibers expressing neonatal MHC were similar in size to control fibers but had significantly fewer myonuclei per millimeter than flight fibers not expressing neonatal MHC. In type I MHC-expressing fibers, the reduction in fiber cross-sectional area was greater than the reduction in myonuclear number; thus the average cytoplasmic volume per myonucleus was significantly lower in flight than in control fibers. The reduction in both myonuclear number and fiber size of fibers expressing type I MHC after 14 days of spaceflight supports the hypothesis that changes in the number of myonuclei may be a contributing factor to the reduction in fiber size associated with chronic unloading of the musculature.