Force characteristics of skeletal muscle of knockout mice lacking creatine (Cr) due to a deletion of guanidinoacetate methyltransferase (GAMT) were studied in situ. Medial gastrocnemius muscles of anesthetized GAMT-deficient (GAMT Ϫ/Ϫ ) and control (Con) littermates were stimulated at optimum length via the sciatic nerve at different stimulation frequencies . GAMT Ϫ/Ϫ mice showed reduced maximal tetanic and twitch force, reduced relative force at 60 Hz, and increased relaxation times. High-intensity fatigue protocols consisting of 30 successive isometric or dynamic contractions showed a strong reduction in force at the beginning of the series in GAMT Ϫ/Ϫ mice, followed by a smaller reduction compared with Con littermates toward the end of the series. Cr supplementation for 2 days in GAMT Ϫ/Ϫ animals (GAMT Cr Ϫ/Ϫ ) resulted in normalization to Con values for relaxation times, relative force at lower stimulation frequencies, and relative force during 30 isometric contractions. Force per muscle mass, however, remained decreased. Furthermore, GAMT Cr Ϫ/Ϫ mice showed differences compared with both Con and unsupplemented animals in maximal rates of force rise and relaxation times during the isometric protocol as well as in force during the dynamic protocol. Our results show that the absence of Cr plays a direct role in relaxation times, maximal rate of force rise, and force production during high-intensity fatigue protocols. The lower force per muscle mass, however, is probably caused by other factors; i.e., high intracellular guanidinoacetate concentrations. energy metabolism; creatine; fatigue; force characteristics THE CREATINE KINASE (CK) reaction, where creatine (Cr) and ATP are reversibly catalyzed to form phosphocreatine (PCr), ADP, and a hydrogen ion, is considered to be important for energy metabolism during muscular exercise (e.g., 15, 32). This so-called PCr-CK system is thought to play a key role in keeping ATP-to-ADP ratios balanced and possibly also in the transport of high-energy phosphates through the cytosol (2,33,35). In skeletal muscle, two major isoforms of CK are present: mouse muscle (MM-CK) in the cytosol and skeletal (ScCKmit) in the intermembrane space of the mitochondria (33).In the past decade, the significance of the PCr-CK system on muscle performance and energy metabolism has been studied in muscles of mice lacking either one or both of the isoforms of CK that are present in muscle (7,9,16,22,25,28,30), and even earlier in rats fed analogs of Cr (e.g