We studied the coordinated regulation of myosin heavy chains (MHC) and metabolic enzymes within individual overloaded adult rat plantaris fibers. This was done using monoclonal antibodies raised against distinct developmental and adult MHCs, and quantitative microphotometric succinate dehydrogenase (SDH) and glycerol-3-phosphate dehydrogenase (GPDH) enzyme assays. Overload shifted MHC expression in the order IIb-->IIx-->IIa-->alpha/I, with a tripling of cells coexpressing I and alpha-MHC, and a transient reexpression of two embryonic MHC and the neonatal isoform in preexisting myofibers. Overload caused a rapid, size-independent, 50% decrease in GPDH activity across all cell types, which recovered by 6 wk. Fiber SDH activities varied according to MHC composition, such that overloaded fibers coexpressing IIa MHC displayed control slow fiber SDH levels, whereas cells expressing IIx and IIb MHC displayed a transient 30% increase in SDH that recovered by 6 wk. Our results suggest that during overload, fibers adapt progressively to the new functional requirements and display more efficient cellular energy utilization and delivery characteristics. The time course of adaptations suggests a role for glycolytic enzymes in the initiation of these transformations.