The purpose of this study was to determine the effect of altering substrate availability on metabolism and performance during intense cycling. Seven highly trained men ingested a random order of three isoenergetic meals 90 min before cycling at 80 % maximal oxygen uptake (VO2max) for 20 min (about 310 W), followed by a 600 kJ time trial lasting about 30 min. Meals consisted of either 1·2 g saturated fat/kg body mass (BM) with 3500 U heparin intravenously (HIFAT) to elevate circulating plasma free fatty acid (FA) concentration, 2·5 g carbohydrate/kg BM (CHO) to elevate plasma glucose and insulin concentrations or 2·5 g carbohydrate+20 mg nicotinic acid/kg BM (NA) to suppress lipolysis and reduce free FA concentration. HIFAT elevated free FA concentration (HIFAT 1·3 (SEM 0·2), CHO 0·2 (sem 0·1), NA 0·1 (sem 0·1) mm; P<0·001), lowered the RER (HIFAT 0·94 (sem 0·01), CHO 0·97 (sem 0·01), NA 0·98 (sem 0·01); P<0·01) and increased the rate of fat oxidation (HIFAT 24 (sem 3), CHO 12 (sem 2), NA 8 (sem 3) μmol/kg per min; P<0·01) during the 20 min ride. Marked differences in fat availability and fuel utilisation, however, had little effect on performance in the subsequent time trial (HIFAT 320 (sem 16), CHO 324 (sem 15), NA 315 (sem 13) W). We conclude: (1) increased fat availability during intense cycling increases the rate of fat oxidation; but (2) the reduction in the rate of carbohydrate oxidation in the presence of high circulating plasma free FA is unlikely to enhance intense exercise performance lasting about 1 h; (3) substrate selection during intense (about 80 % VO2max) exercise is dominated by carbohydrate oxidation.