This study examined the effect of high vs moderate exercise intensity on changes in concentration of plasma pyridoxal 5'-phosphate (PLP), pyridoxal (PL), and 4-pyridoxic acid (4-PA) in human subjects. Eight physically active subjects were tested twice at 60% and 85% maximum oxygen consumption (VO2max) for 30 and 20 min, respectively, on a bicycle ergometer. Blood samples were obtained before and during exercise. Data were adjusted for changes in plasma volume calculated from changes in hematocrit. PLP concentrations significantly increased during exercise (P < 0.0001), with 79% of the rise in PLP concentration occurring within 5 min. 4-PA concentration increased steadily through exercise and was 23% higher at 20 min than at 0 min. Exercise intensity had no effect on the magnitude or rate of either increase for either PLP or 4-PA. PL concentration did not vary with exercise duration, but was significantly higher at 60% compared with 85% VO2max (P = 0.001). No significant differences were observed in glucose concentration. The data do not support hypotheses in the literature that PLP concentration rises during exercise to support exercise-induced shifts in substrate utilization.