We compared several sets of conditions used to estimate metabolism in rat lung slices. 14CO2 production from [14C]glucose, oxygen consumption, lactate production, and glucose consumption were used as measures of metabolic activity. The calculated results differed when we used 1) different techniques for estimating tissue weight, 2) tissue slices of 0.3-, 0.5-, 0.7-, and 1.0-mm thickness, 3) 95% air or 95% oxygen with 5% CO2 4) a delay after slice preparation and 4 degrees C and room temperature or periods of anoxia before incubation, 5) shaking rates of 60, 90, 120, or 150/min, 6) phosphate or bicarbonate buffers. Conditions of maximal activity were found using 95% O2 with 1.0-mm tissue slices, shaking at 120/min in phosphate buffer without periods of hypoxia or undue delays before incubation. Tissue weight should be obtained without exposure to aqueous solutions or dehydration by contact with cotton gauze or filter paper.