The influence of microgravity on the myosin phenotype of skeletal muscle fibers in the vastus lateralis of eight crew members was studied before and after 5-day (n = 3) and 11-day (n = 5) spaceflights (space shuttle flights: STS-32, -33 and -34). Single-fiber electrophoresis analyses showed that the proportion of fibers expressing only slow (type I) myosin heavy chain (MHC) in the vastus lateralis was significantly lower after than before 11 days of spaceflight. Although the family of type II MHC isoforms was elevated post- compared with preflight, the distribution among the isoforms of type II MHC was not statistically different. Based on monoclonal and polyclonal antibodies specific for three adult MHC isoforms and single-fiber electrophoresis, approximately 3% of the fibers analyzed coexpressed all three adult MHC isoforms. The results from immunohistochemical staining with two different sets of antibodies indicate a reduction in the percentage of fibers expressing type I MHC as a result of spaceflight. The mean difference, however, was significant only when the fibers were categorized simply as type I or II. These changes appeared to be highly individualized among the astronauts. These results suggest that a rapid change in MHC isoform expression can occur in some muscle fibers after a relatively brief exposure to spaceflight.