The effects of correct alignment of the ventilation and fractional gas concentration during breath-by-breath calculation of oxygen uptake (VO2) have been examined during exercise in four subjects, at each of 50, 100, 150, and 200 W. Data were analyzed using lagtimes in the range of 200 to 400 msec to align ventilation with gas fraction. VO2 was markedly affected by lagtime. Calculated values of VO2 at 200 W ranged from 1953 +/- 55 (mean +/- SEM), to 2583 +/- 29, to 2843 +/- 28 ml.min-1 with lagtimes of 200, 310, and 400 msec, respectively. Mean values from a mixing box system did not differ significantly from the mean of the breath-by-breath data collection with a lagtime of 310 msec. Additional computations have shown that temperature correction can markedly affect calculated ventilatory volumes and N2 balance. VO2 was not changed because of the compensation from the calculated effects of changes in lung gas stores.