Prolonged exercise followed by a brief period of reduced activity has been shown to result in an overshoot in maximal sarcoplasmic reticulum (SR) Ca2+-ATPase activity [maximal velocity ( V max)] in rat locomoter muscles (Ferrington DA, Reijneveld JC, Bär PR, and Bigelow DJ. Biochim Biophys Acta 1279: 203–213, 1996). To investigate the functional significance and underlying mechanisms for the increase in V max, we analyzed Ca2+-ATPase activity and Ca2+ uptake in SR vesicles from the fast rat gastrocnemius muscles after prolonged running (RUN) and after prolonged running plus 45 min of low-intensity activity (RUN+) or no activity (REC45) and compared them with controls (Con). Although no differences were observed between RUN and Con, both V max and Ca2+ uptake were higher ( P < 0.05) by 43 and 63%, respectively, in RUN+ and by 35 and 34%, respectively, in REC45. The increase in V max was accompanied by increases ( P < 0.05) in the phosphorylated enzyme intermediate measured by [γ-32P]ATP. No differences between groups for each condition were found for the fluorescent probes FITC and ( N-cyclohexyl- N 1-dimethylamino-α-naphthyl)carbodiimide, competitive inhibitors of the nucleotide-binding and Ca2+-binding sites on the enzyme, respectively. Similarly, no differences for the Ca2+-ATPase were observed between groups in nitrotyrosine and phosphoserine residues, a measure of nitrosylation and phosphorylation states, respectively. Western blots indicated no changes in relative isoform content of sarcoendoplasmic reticulum (SERCA)1 and SERCA2a. It is concluded that the increase in V max of the Ca2+-ATPase observed in recovery is not the result of changes in enzyme nitroslyation or phosphorylation, changes in ATP and Ca2+-binding affinity, or changes in protein content of the Ca2+-ATPase.