To test the validity of cotton balls as a urine collection medium for hydrogen and oxygen isotope ratio measurements, we moistened cotton balls with 1-, 5-, and 10-mL aliquots of urine that contained either natural abundances or one of two enriched levels of 2H and 18O. The liquids were then expressed from the cotton balls, either at once, or after 30 or 60 min of atmospheric exposure, and analyzed for their 2H/1H and 18O/16O isotope ratios. The 2H abundances of the fluids expressed from the cotton balls were significantly lower than the original values. This dilution of 2H, however, diminished in the samples that had a greater volume of urine. We observed no effects of volume or time on 18O. Our results indicate that at low urine volumes, ambient moisture dilutes 2H in the cotton balls, but isotope exchanges and evaporation have little or no effect on the hydrogen and oxygen isotopic abundances of the urine samples. Total body water and energy expenditure values calculated from the 2H and 18O enrichments of 5- and 10-mL urine samples were within 1% of the theoretical values. Therefore, cotton balls are suitable for collection of infant urine samples for hydrogen and oxygen isotope ratio measurements, if the volume of urine that can be expressed from a cotton ball is > or = 5 mL.