Glycerinated rabbit psoas fibers were tested for their ability to contract under the influence of creatine phosphate and creatine kinase in the absence of free nucleotide. Tension development by the fibers was observed upon addition of creatine phosphate to the medium containing creatine kinase purified to the first lyophilization stage. However, when the enzyme was washed free of nucleotides by treatment with the anion exchange resin Dowex 1, no contraction occurred until free nucleotide was supplied. In all experiments, contractile activity of the psoas fibers was the criterion for determining the enzyme activity concerned. Using this criterion, creatine kinase activity native to the glycerinated fibers was also demonstrated. No evidence for direct transphosphorylation of the bound nucleotide of the fiber was found.