We studied the effects of 10 wk of functional overload on the expression of myosin heavy chain (MHC), sarcoplasmic reticulum Ca²⁺-ATPase isoforms (SERCA), and the activity of several metabolic enzymes in sham and regenerated plantaris muscles. Overload was accomplished by bilateral surgical ablation of its synergists 4 wk after right plantaris muscles regenerated after myotoxic infiltration. The overload-induced muscle enlargement was slightly less in regenerated than in sham muscles [28% (P < 0.005) and 43% (P < 0.001), respectively]. Overload led to an increase in type I MHC expression (P < 0.01) to a similar extent in sham and regenerated plantaris, while the expected shift from type IIb to type IIa MHC was less marked in regenerated than in sham plantaris. The overload-induced decrease in the expression of the fast SERCA isoform and in the activity of the M subunit of lactate dehydrogenase occurred to a similar extent in sham and regenerated plantaris [66% (P < 0.01) and 27% (P < 0.005), respectively]. In conclusion, the lesser responses of muscle mass and fast MHC composition of regenerated plantaris to mechanical overload suggest an alteration of the transcriptional, translational, and/or posttranslational control of gene expression in regenerated muscle.