This study investigates the surface activity of rabbit pulmonary surfactant subfractions at different concentrations in a new pressure-driven captive bubble surfactometer, which provides more rigorous testing conditions than heretofore applied to this material. Subfractions were separated by centrifugation of lavage return into a third (P3; 1,000 x average g, 20 min), a fourth (P4; 60,000 x average g, 60 min), and a fifth (P5; 100,000 x average g, 16 h) pellet. At 2.0 mg phospholipid/ml, P3 and P4 demonstrated more rapid adsorption, lower minimum surface tensions on first and subsequent compressions, and lower monolayer compressibilities than did P5. This surface activity of P3 and P4 increased with concentration between 0.1 and 2.0 mg phospholipid/ml. Adsorption rate constants were approximately 10,000 times faster than desorption constants. We conclude that, in a normal lung, components of P3 and P4 determine alveolar surface tension. We speculate that under special circumstances even the less surface active P5 could have an important influence at the air-water interface.