The phosphoarginine shuttle system effectively regenerates ATP in the cilia of Paramecium caudatum. To estimate the effective concentration of ATP-regenerating enzymes, we attempted to reconstitute certain ATP-regenerating systems within the cilia of intact cortical sheets using exogenous enzymes and high-energy substances. The addition of phosphoenolpyruvate, which is one of the substrates in glycolysis, did not increase the ciliary beat frequency, whereas phosphocreatine together with exogenous creatine kinase, effectively increased the ciliary beat frequency. In the presence of 0.6 similar to mg/ml creatine kinase and 0.4 similar to mM phosphocreatine, the ciliary beat frequency was comparable to that produced by the addition of phosphoarginine. This result indicates that the reconstituted phosphocreatine shuttle system can work as an artificial ATP-regenerating system for ciliary movements. The effective concentration of creatine kinase in the reconstituted phosphocreatine shuttle system was estimated to be about 7.4 similar to mu M based on the molecular mass of creatine kinase (MW 81,000). Therefore, the effective concentration of arginine kinase in the cilia of live Paramecium is approximately 10 similar to mu M. This estimated concentration of intraciliary arginine kinase is sufficient to maintain a high ATP concentration throughout the cilia of P. caudatum.