Nitric oxide (NO) generated from NO synthases mediates normal skeletal muscle functions. Biosynthesis of NO apparently is linked to muscle activity, but the distribution and expression of the three major NO synthase 1-3 isoforms under conditions of extended muscle disuse and exercise are still unclear. Our aim was to elucidate whether protein levels and the cellular or subcellular localization patterns of NO synthases underwent significant changes in a mixed fast/slow and slow type skeletal muscle after prolonged disuse in a long-term bed rest study, a useful experimental paradigm of simulated microgravity in ground-based space research. We examined whether resistance exercise performed regularly as a countermeasure to progressive atrophy within 12 wk of strict bed rest would support expression of one or more isoforms of NOS, thereby maintaining normal skeletal muscle functions during immobilization in clinical settings or in human spaceflight.