The quantitation of substances in the epithelial lining fluid (ELF) of the lower respiratory tract, as obtained by bronchoalveolar lavage (BAL), is not precise because of the variable dilution of the ELF by the instilled lavage fluid. It has been reported that the absolute concentration of proteins in ELF can be determined by using the ratio of urea concentration in BAL fluid to that in serum as a method to calculate the volume of ELF recovered by BAL. Furthermore, it has been suggested that the error caused by diffusion of urea into the instilled lavage fluid can be minimized by instilling only 100 ml (5 × 20 ml) of saline rather than 300 ml (6 × 50 ml). We tested the validity of this method by collecting and individually analyzing aliquots from 2 different BAL protocols—a 100-ml (5 × 20 ml) BAL and a 300-ml (6 × 50 ml) BAL— performed in 6 healthy, nonsmoking subjects. Total protein, albumin, and urea were measured in each aliquot and in pooled fluid from each BAL procedure, and urea was measured in serum. In the 300-ml BAL, total protein and albumin concentrations tended to decrease progressively from the second to the sixth aliquots. In contrast, the urea concentration increased progressively from the first to the sixth aliquots. The concentration of albumin in ELF, calculated from the concentration of urea and albumin in each BAL aliquot, tended to decrease in each successive aliquot, becoming significant by the fourth aliquot. In the 100-ml BAL, total protein and albumin did not change significantly in successive aliquots, but the urea concentration increased from the first to the fifth aliquot. The concentration of albumin in ELF, calculated in each BAL aliquot in the 100-ml BAL, tended to decrease from the second to the fifth aliquot, but this did not reach statistical significance. These observations suggest that a significant amount of urea diffuses into the instilled BAL fluid as it dwells in the alveolar space during both the 100-ml and the 300-ml BAL protocols. This diffusion of urea will cause errors in the calculated ELF recovery despite the use of a smaller volume of lavage fluid.