The effects of short-term (4 days) and long-term (60 days) neuromuscular inactivity on myonuclear number, size and myosin heavy chain (MHC) composition of isolated rat soleus fibers were determined using confocal microscopy and gel electrophoresis. Inactivity was produced via spinal cord isolation (SI), i.e., complete spinal cord transections at a mid-thoracic and a high sacral level and bilateral deafferentation between the transection sites. Compared to control, there was an increase in the percentage of fibers containing the faster MHC isoforms after 60, but not 4, days of SI. The mean sizes of type I and type I+IIa fibers were 41 and 27% and 66 and 56% smaller after 4 and 60 days of SI, respectively. Thus, atrophy occurred earlier than the shift in MHC profile. The number of myonuclei was ~30% higher in type I than type I+IIa fibers in control soleus, but after 60 days of SI these values were similar. The number of myonuclei/mm in type I fibers was significantly lower than control after 60 days of SI, whereas there was no change in type I+IIa fibers. Thus, myonuclei were eliminated from fibers containing only type I MHC. Since the magnitude of the loss of myonuclei was less than the level of atrophy, the myonuclear domains of both type I and type I+IIa fibers were significantly lower than control. Thus, chronic (60 days) inactivity results in smaller, faster fibers that contain a higher than normal amount of DNA per unit of cytoplasm. The absence of activation of muscle fibers that are normally the most active (pure type I fibers) resulted in most, but not all, fibers expressing some fast MHC isoforms. The results also indicate that a loss of myonuclei is not a prerequisite for sustained muscle fiber atrophy.