Rat fast-twitch muscles were tetanically stimulated in situ with an occluded circulation to examine ATP utilization and provision during isometric tension production. Plantaris (PL) and gastrocnemius (G) muscles were stimulated for 60 s in four conditions: A) 1.0-Hz train rate, 200-ms train duration at 80 Hz, B) 1.0 Hz (100 ms, 80 Hz), C) 0.5 Hz (100 ms, 80 Hz), and D) 1.0 Hz (200 ms, 40 Hz). Muscles were sampled pre- and post-stimulation for pH, high-energy phosphates, and glycolytic intermediates. Contributions to total ATP utilization (all muscles and conditions) were 64-67% glycolysis, 24-28% phosphocreatine, and 8-9% endogenous ATP. Glycogenolysis and glycolysis were greatest in white G (WG), 40% lower in red G (RG), and intermediate in PL muscles. Average energy costs in conditions A and D were approximately 0.60 mumol ATP/(N.s). Decreasing the train duration to 100 ms in B and the number of tetani to 30 in C increased energy costs to 0.93 +/- 0.05 and 1.26 +/- 0.07 mumol ATP/(N.s). Despite a lower pH, WG glycogenolytic (phosphorylase) activity was constant during condition A, whereas RG activity decreased in the final 30 contractions. Larger accumulations of Pi and inosine monophosphate may account for the maintained phosphorylase activity. Glycolytic (phosphofructokinase, PFK) activity was highest in WG and associated with higher fructose 6-phosphate concentration, greater depletion of ATP and, in later contractions, a higher NH4+ concentration. During tetanic in situ stimulation of fast-twitch muscle, the H+ profiles of phosphorylase and PFK are extended beyond in vitro predictions via the accumulation of positive modulators. This permits significant anaerobic ATP production via the glycolytic pathway despite increasing [H+]. The findings also suggest that lengthening the duration of tetani, generating lower peak tensions, and prolonging relaxation time all contribute to lower energy costs in fast-twitch muscle.