Abstract  This study investigated the influence of environmental heat stress on ammonia (NH₃) accumulation in relation to nucleotide metabolism and fatigue during intermittent exercise. Eight males performed 40 min of intermittent exercise (15 s at 306±22 W alternating with 15 s of unloaded cycling) followed by five 15 s all-out sprints. Control trials were conducted in a 20°C environment while heat stress trials were performed at an ambient temperature of 40°C. Muscle biopsies and venous blood samples were obtained at rest, after 40 min of exercise and following the maximal sprints. Following exercise with heat stress, the core and muscle temperatures peaked at 39.5±0.2 and 40.2±0.2°C to be ~ 1°C higher (P<0.05) than the corresponding control values. Mean power output during the five maximal sprints was reduced from 618±12 W in control to 558±14 W during the heat stress trial (P<0.05). During the hot trial, plasma NH₃ increased from 31±2 µM at rest to 93±6 at 40 min and 151±15 µM after the maximal sprints to be 34% higher than control (P<0.05). In contrast, plasma K⁺ and muscle H⁺ accumulation were lower (P<0.05) following the maximal sprints with heat stress compared to control, while muscle glycogen, CP, ATP and IMP levels were similar across trials. In conclusion, altered levels of "classical peripheral fatiguing agents" does apparently not explain the reduced capacity for performing repeated sprints following intermittent exercise in the heat, whereas the augmented systemic NH₃ response may be a factor influencing fatigue during exercise with superimposed heat stress.