Functional compartmentation and metabolism of radioactive proline was evaluated to define conditions under which synthesis of lung proteins could be measured accurately based on proline incorporation. Rat lungs were perfused with Krebs-Henseleit bicarbonate buffer equilibrated with O2/N2/CO2 (20:75:5) and containing 4.5% (w/v) bovine serum albumin, 5.6 mM glucose and amino acids at plasma levels. Intracellular proline increased linearly as perfusate proline concentration was increased from 108 microM, the plasma level, to 540 or 1080 microM. At each concentration, the pool of proline which provided precursors to protein synthesis rapidly reached a steady-state specific radioactivity, but when extracellular proline was 108 microM, this pool was diluted significantly by proline from endogenous sources. At 540 or 1080 microM extracellular proline, the specific radioactivities of perfusate and intracellular proline approached equality and rates of protein synthesis calculated based on the specific radioactivity of extracellular proline compared favorably with those calculated from the specific radioactivity of phenylalanyl-tRNA. Similar results were obtained in lungs of two groups of rats in which intracellular proline concentration differed 3-fold. Thus, the contribution of endogenous proline to the pathway of protein synthesis was minimized when extracellular proline was present at high concentration. Under this condition, calculations of protein synthesis based on proline incorporation were most accurate.