Retention of radiolabeled phospholipid vesicles instilled into the alveolar space was studied with the isolated perfused rat lung and quantitated by measuring the percent of instilled radioactivity remaining in lung after five lavages. With synthetic [14C]dipalmitoyl phosphatidylcholine (PC):egg PC: phosphatidylglycerol:cholesterol (10:5:2:3) vesicles, there was a rapid (within 5 min) base-line retention of 10.3 +/- 0.25% (n = 11) followed by a slower phase of linear retention over the next 4 h. Retention at 2 h with perfused lungs was 18.6 +/- 0.60% (n = 9) and was similar to values obtained with lungs in vivo. Net retention (total minus base line) was stimulated 93% by isoproterenol, 173% by 8-bromoadenosine 3′,5′-cyclic monophosphate (8-Br-cAMP), and 39% by 8-bromoguanosine 3′,5′-cyclic monophosphate; propranolol blocked the effect of isoproterenol. The retention of natural (biosynthesized) surfactant [14C]PC was stimulated 92% by 8-Br-cAMP. The results suggest that the retention of exogenous phospholipid by the isolated perfused lung represents phospholipid uptake and that this process is under beta-adrenergic control. Secretion and uptake may be physiologically linked to regulate the concentration of surfactant on the alveolar surface.