Measurements of pulmonary carbon monoxide (CO) excretion can be used as a quantitative indicator of heme turnover. We determined whether a diurnal variation of heme turnover occurs by using a recently described technique that estimates breath CO excretion from measurements of alveolar CO concentration corrected for environmental CO to yield an endogenous PCO. This simple technique, unlike the previously employed rebreathing method, makes it possible to repeatedly measure CO excretion throughout a 24-hour period. Nine studies in seven healthy adult subjects demonstrated a diurnal rhythm of CO excretion with a peak excretion rate at about noon that was 26% greater than the nadir, which occurred at about midnight. The rhythm of CO production underlying the observed breath CO excretion was calculated to have an amplitude of about twice that of CO excretion and a phase shift relative to excretion of about 4 hours. We conclude that a diurnal variation in the rate of heme turnover occurs, and when CO determinations are used to assess minor alterations in heme turnover, consideration must be given to the time of day at which the measurements are obtained.