We have determined the effect of pulsatile flow on segmental vascular resistance in lungs from 29 adult rabbits. In group I (n = 4), II (n = 8), and III (n = 8) lungs were isolated. In group IV (n = 9) rabbits were anesthetized, their chests were opened, and lungs were studied in vivo. Group I and II lungs had steady-flow perfusion: group I with intact vasotonus and group II with papaverine treatment. Group III lungs (papaverine treated) were perfused for two consecutive 45-min periods with steady and pulsatile flow. In all isolated lungs and in lungs of five anesthetized rabbits, we measured pressures in subpleural 20- to 50-microns-diam arterioles and venules by use of the micropipette servo-nulling method. Measurement of distribution of blood flow in lungs of four anesthetized rabbits by use of radiolabeled microspheres revealed no abnormality of blood flow to the micropunctured lobe. We found that total and segmental vascular resistances were similar in group I and II lungs, with microvessels representing 55% of total resistance. In group III lungs, total resistance was 30% lower during pulsatile flow than during steady flow because of a lower microvascular resistance. Lungs in vivo (group IV) had a significantly lower total vascular resistance than isolated lungs and had a low fractional resistance in microvessels (approximately 28%). We conclude that, in isolated perfused adult rabbit lungs, vascular resistance is very high, particularly in the microvascular segment, and that pulsatile flow decreases microvascular resistance.