In the doubly labeled water (DLW) method, for measurement of energy expenditure in humans, calculation of carbon dioxide production rate is influenced by the ratio (R) of the dilution spaces of the tracer isotopes 2H and 18O. Recent publications have recommended use of a fixed value for R, using a mean population value (meanR) rather than individually derived experimental values. However, most of the data considered previously were derived from adults. Infants are characterized by rapid rates of energy deposition and high body water content, either of which could influence isotope kinetics. We have considered whether use of a fixed R value is suitable for DLW studies of infants, and whether the adult (meanR) value is appropriate. Data from 281 DLW measurements in healthy infants aged 6 wk to 12 mo were analyzed. Mean R of all measurements was 1.036 (SD 0.019), very similar to (meanR) values recommended for adults. We found analytical precision could account for almost all between-subject variation at a single time point. However, we also found relationships between R and age and weight gain, indicating that physiology does influence the space ratio. The effect of weight gain on R was negligible, but changes of (meanR) with age, reflecting changes in percentage body water during infancy, were of sufficient magnitude to affect calculation of energy expenditure significantly. Our analysis suggests that use of a fixed ratio at a single time point in infancy is acceptable, but that the appropriate value of (meanR) changes over the first year of life.