To investigate the differential contribution of oxidative and substrate-level phosphorylation to force production during repetitive, maximal tetanic contractions, single skeletal muscle fiber performance was examined under conditions of high-oxygen availability and anoxia. Tetanic force development (P) was measured in isolated, single type-1 muscle fibers (fast twitch; n = 6) dissected from Xenopus lumbrical muscle while being stimulated at increasing frequencies (0.25, 0.33, and 0.5 Hz), with each frequency lasting 2 min. Two separate work bouts were conducted, with the perfusate PO₂ being either 0 or 159 mmHg. No significant (P < 0.05) difference was found in the initial peak tensions (P₀) between the high (334 ± 57 kPa) and the low (325 ± 41 kPa) PO₂ treatment. No significant difference in P was observed between the treatments during the first 50 s. However, a significant difference in force production was observed between the high (P/P₀ = 0.96 ± 0.02) and the low PO₂ condition (P/P₀ = 0.92 ± 0.02) by 60 s of work. After 60 s, steady-state force production was maintained during the high compared with the low PO₂ condition until stimulation frequency was increased, at which point developed tension during the high PO₂ condition began to decline. Time to fatigue (P/P₀ = 0.3) was reached significantly sooner during the low (250 ± 16 s) than the high PO₂ condition (367 ± 28 s). These results demonstrate that during the first 50 s of 0.25-Hz contractions, substrate-level phosphorylation has the capacity to maintain force and ATP hydrolysis when oxidative phosphorylation is absent. This period was followed by an oxygen-dependent phase in which force generation was maintained during the high PO₂ condition (but not during the low PO₂ condition) until the onset of a final fatiguing phase, at which a calculated maximal rate of oxidative phosphorylation was reached.