In this study we examined the time course of changes in the plasma concentration of oxypurines [hypoxanthine (Hx), xanthine and urate] during prolonged cycling to fatigue. Ten subjects with an estimated maximum oxygen uptake ( V?O 2max) of 54 (range 47–67) ml?·?kg -1?·?min -1 cycled at [mean?(SEM)] 74?(2)% of V?O 2max until fatigue [79?(8) min]. Plasma levels of oxypurines increased during exercise, but the magnitude and the time course varied considerably between subjects. The plasma concentration of Hx ([Hx]) was 1.3?(0.3)?µmol/l at rest and increased eight fold at fatigue. After 60?min of exercise plasma [Hx] was >10?µmol/l in four subjects, whereas in the remaining five subjects it was <5?µmol/l. The muscle contents of total adenine nucleotides (TAN?=?ATP+ADP+AMP) and inosine monophosphate (IMP) were measured before and after exercise in five subjects. Subjects with a high plasma [Hx] at fatigue also demonstrated a pronounced decrease in muscle TAN and increase in IMP. Plasma [Hx] after 60?min of exercise correlated significantly with plasma concentration of ammonia ([NH 3], r?=?0.90) and blood lactate ( r?=?0.66). Endurance, measured as time to fatigue, was inversely correlated to plasma [Hx] at 60?min ( r?=?-0.68, P?3] or blood lactate. It is concluded that during moderate-intensity exercise, plasma [Hx] increases, but to a variable extent between subjects. The present data suggest that plasma [Hx] is a marker of adenine nucleotide degradation and energetic stress during exercise. The potential use of plasma [Hx] to assess training status and to identify overtraining deserves further attention.