Effects of gravitational loading or unloading on the gain of the characteristics in soleus muscle fibers were studied in rats. The tail suspension was performed in newborn rats from postnatal day 4 to month 3, and the reloading was allowed for 3 mo in some rats. Single expression of type I myosin heavy chain (MHC) was observed in approximately 82% of fibers in 3-mo-old controls, but the fibers expressing multiple MHC isoforms were noted in the unloaded rats. Although 97% of fibers in 3-mo-old controls had a single neuromuscular junction at the central region of fiber, fibers with multiple nerve endplates were seen in the unloaded group. Faster contraction speed and lower maximal tension development, even after normalization with fiber size, were observed in the unloaded pure type I MHC fibers. These parameters generally returned to the age-matched control levels after reloading. It was suggested that antigravity-related tonic activity plays an important role in the gain of single neural innervation and of slow contractile properties and phenotype in soleus muscle fibers.