Using an immunohistochemical double-labeling technique, we observed that different isoforms of sarcoplasmic reticulum Ca-ATPase are co-expressed in single fibers of canine fast-twitch skeletal muscles stimulated chronically at low frequency. By 7 days of neuromuscular stimulation, the population of hybrid fibers expressing both SERCA1 and SERCA2a [fast- and slow-twitch isoforms of sarco(endo)plasmic reticulum Ca(2+)-ATPase] had increased from 1.5% to 9.2% of fibers. By 14 days of stimulation 90% of the pure fast-twitch fibers (expressing only SERCA1) were replaced by hybrid fibers. An additional 28 days of stimulation caused all fast-twitch fibers to express SERCA2a at the same level as found in nonstimulated slow-twitch fibers (expressing only SERCA2a). At this time, one-half of the previously hybrid fibers had become pure-slow-twitch fibers. The remaining one-half of the hybrid fibers expressed SERCA1 at a very low level. Extending stimulation to 70 days did not further change the percentage of fibers that were slow-twitch or hybrid. Immunoblot studies at the whole-muscle level confirmed that changes in SERCA expression at 42 days of neuromuscular stimulation were complete. Immunohistochemical analysis of longitudinal sections of muscle showed that the changes in SERCA protein were uniform along the length of the muscle fiber, indicating that nuclei along its length responded equally to chronic stimulation.