The aim of this study was to evaluate myofibrillar creatine kinase (EC 2.7.3.2) activity on the background of the effect of substrate channeling by myosin ATPase and to compare it with creatine kinase (CK) activity of whole skinned fibers. In order to assess CK activity, skinned fibers were prepared from the rat psoas major muscles defined by light microscopy. The activity in permeabilized fibers after treatment with saponin, Triton X-100 and Ca2+-free medium reached 2.80, 6.97 and 3.32 m mol ATP min-1 mg-1 protein, respectively, when a coupled enzyme assay system with external hexokinase and glucose-6-phosphate dehydrogenase was used. Transmission electron microscopy (TEM) revealed a possible interference among activities of sarcolemmal, sarcoplasmic, myofibrillar and mitochondrial CK from persisting structures. For evaluation of the myofibrillar CK itself, a pure myofibrillar fraction was prepared. Fraction purity was confirmed by TEM and by enzymatic assays for marker enzymes. Two procedures, i.e. the coupled enzyme assay and the evaluation of phosphocreatine (Pcr) concentration before and after the CK reaction, were used for measurement of CK activity in this fraction. The procedures resulted in 3.2 nmol ATP min-1 mg-1 protein and 7.6 nmol PCr min-1 mg-1 protein, respectively. These alternative approaches revealed a discrepancy between the reacting portions of Pcr by more than 50 % , which provides information about the size of the effect, generally described as substrate channeling., M. Gregor, J. Mejsnar, A. Janovská, J. Žurmanová, O. Benada, B. Mejsnarová., and Obsahuje bibliografii