This study investigated the effects of acute creatine kinase (CK) inhibition (CKi) on contractile performance, cytosolic Ca2+ concentration ([Ca2+]c), and intracellular PO2 (PIO2) in Xenopus laevis isolated myocytes during a 2-min bout of isometric tetanic contractions (0.33-Hz frequency). Peak tension was similar between trials during the first contraction but was significantly (P < 0.05) attenuated for all subsequent contractions in CKi vs. control (Con). The fall in PIO2 (DeltaPIO2) from resting values was significantly greater in Con (26.0 +/- 2.2 Torr) compared with CKi (17.8 +/- 1.8 Torr). However, the ratios of Con to CKi end-peak tension (1.53 +/- 0.11) and DeltaPO2 (1.49 +/- 0.11) were similar, suggesting an unaltered aerobic cost of contractions. Additionally, the mean response time (MRT) of DeltaPIO2was significantly faster in CKi vs. Con during both the onset (31.8 +/- 5.5 vs. 49.3 +/- 5.7 s; P < 0.05) and cessation (21.2 +/- 4.1 vs. 68.0 +/- 3.2 s; P < 0.001) of contractions. These data demonstrate that initial phosphocreatine hydrolysis in single skeletal muscle fibers is crucial for maintenance of sarcoplasmic reticulum Ca2+ release and peak tension during a bout of repetitive tetanic contractions. Furthermore, as PIO2 fell more rapidly at contraction onset in CKi compared with Con, these data suggest that CK activity temporally buffers the initial ATP-to-ADP concentration ratio at the transition to an augmented energetic demand, thereby slowing the initial mitochondrial activation by mitigating the energetic control signal (i.e., ADP concentration, phosphorylation potential, etc.) between sites of ATP supply and demand.