In order to determine the stability of directly measured pH and Pco<sub>2</sub>, and calculated [HCO<sub>3</sub>] in stored urine, 11 alkali-loaded normal subjects provided 33 spot and sixteen 24-hour mineral oil-covered, thymol-preserved, refrigerated urine samples. For the spot samples, pH and Pco<sub>2</sub> were measured immediately and again at 4 and 24 h. In addition, immediately after voiding, 24 of the spot samples were split into oil-covered and no-oil moieties and analyzed immediately and again at 4 and 24 h. pH and Pco<sub>2</sub> measurements of the 24-hour collections were carried out immediately after completion and again 24 h later. The results demonstrated the importance of using oil to limit the escape of CO<sub>2</sub> from stored urine. Thus, after 24 h the oil-uncovered subgroup of 24 spot urine samples sustained a 82% decline in Pco<sub>2</sub> and a 20% fall in [HCO<sub>3</sub>]. In contrast, the corresponding percentage decrements in the oil-covered samples were 16 and 1%, respectively. The results also indicated that even with oil there is loss of CO<sub>2</sub>, which increases with time and which shows a statistically significant direct correlation with the baseline level of Pco<sub>2</sub>· Nevertheless, modest loss of CO<sub>2</sub> usually produces only slight decrements in [HCO<sub>3</sub>] because of the countervailing influence of the resultant increase in pH. We conclude that the use of mineral oil is necessary in order to obtain adequate stability of Pco<sub>2</sub>, pH and [HCO<sub>3</sub>] in refrigerated urine requiring several hours or more of storage.