We examined the effects of exercise intensity and a 10-wk cycle ergometer training program [5 days/wk, 1 h, 75% peak oxygen consumption (VO2 peak)] on plasma free fatty acid (FFA) flux, total fat oxidation, and whole body lipolysis in healthy male subjects (n = 10; age = 25.6 +/- 1.0 yr). Two pretraining trials (45 and 65% of VO2 peak) and two posttraining trials (same absolute workload, 65% of old VO2 peak; and same relative workload, 65% of new VO2 peak) were performed by using an infusion of [1-13C]palmitate and [1,1,2,3, 3-2H]glycerol. An additional nine subjects (age 25.4 +/- 0.8 yr) were treated similarly but were infused with [1,1,2,3,3-2H]glycerol and not [1-13C]palmitate. Subjects were studied postabsorptive for 90 min of rest and 1 h of cycling exercise. After training, subjects increased VO2 peak by 9.4 +/- 1.4%. Pretraining, plasma FFA kinetics were inversely related to exercise intensity with rates of appearance (Ra) and disappearance (Rd) being significantly higher at 45 than at 65% VO2 peak (Ra: 8.14 +/- 1.28 vs. 6.64 +/- 0.46, Rd: 8. 03 +/- 1.28 vs. 6.42 +/- 0.41 mol. kg-1. min-1) (P </= 0.05). After training, when measured at the same absolute and relative intensities, FFA Ra increased to 8.84 +/- 1.1, 8.44 +/- 1.1 and Rd to 8.82 +/- 1.1, 8.35 +/- 1.1 mol. kg-1. min-1, respectively (P </= 0.05). Total fat oxidation determined from respiratory exchange ratio was elevated during exercise compared with rest, but did not differ among the four conditions. Glycerol Ra was elevated during exercise compared with rest but did not demonstrate significant intensity or training effects during exercise. Thus, in young men, plasma FFA flux is increased during exercise after endurance training, but total fat oxidation and whole-body lipolysis are unaffected when measured at the same absolute or relative exercise intensities.