To examine the effects of a dilutional mediated decrease in arterial O2 content on muscle metabolic and substrate behaviour during exercise, plasma volume was acutely expanded by either 14% (LOW) or 21% (HIGH) using a 6% dextran solution dissolved in saline (Macrodex) and compared with a control (CON) condition. The exercise protocol, performed by eight untrained males (VO2max = 45.2 +/- 2.2 mL.kg-1.min-1, X +/- SE) and with the conditions randomized, was conducted for 120 min at 46 +/- 4% VO2max. The content of inosine monophosphate determined on muscle tissue extracted from the vastus lateralis increased (p < 0.05) by 120 min of exercise (0.119 +/- 0.02 vs 0.493 +/- 0.19 mmol/kg dry weight) in CON. No effect of either LOW or HIGH expansion of plasma volume was found. Similarly, phosphocreatine content (mmol/kg dry weight), although reduced (p < 0.05) with exercise, was not different between the conditions at either 3 min (61.9 +/- 3.5, 66.2 +/- 3.5, 64.3 +/- 2.1) or 120 min (52.5 +/- 6.3, 53.8 +/- 5.8, 59.4 +/- 5.5) of exercise. In contrast, both pyruvate and lactate were reduced (p < 0.05) by 3 min of exercise in both LOW and HIGH compared with CON. The reduction in these metabolites with plasma volume expansion was not accompanied by an alteration in glycogen depletion rates. Steady-state VO2 was unaffected by acute hypervolemia. These results suggest that moderate exercise following an approximate 10% reduction in arterial O2 content can be performed without increasing the imbalance between ATP production and utilization rates. Since high energy phosphate transfer and glycolysis appeared not to be increased, mitochondrial respiration was apparently preserved by mechanisms as yet undetermined.