The uptake and distribution of two surfactant mixtures, Exosurf and Infasurf, instilled into the lungs of normal and hyperoxia-exposed (100% O2, 60 h) rabbits, were quantified at the alveolar level using flow cytometry and fluorescence microscopy. The surfactants were labeled with the fluorescent phospholipid analog NBD-C12-PC (1-palmitoyl-2-[12-[(7-nitro-2-1,3-benzoxadiazol-4-yl)amino]dodecanoyl]-sn- glycero-3-phosphocholine) in 100:1 molar ratio. Rabbits were killed 2 h after the instillation of either surfactant (80 mg phospholipid [PL]/kg), and alveolar macrophages (AM) and alveolar type II (ATII) cells were isolated and examined for the presence of NBD fluorescence. The fractions of cells, with NBD fluorescence values higher than autofluorescence, isolated from the lungs of air-breathing rabbits instilled with either Infasurf or Exosurf, were 84% and 63% for AM, and 55% and 45% for ATII cells. Exposure of rabbits to hyperoxia decreased the fraction of NBD-positive AM following Infasurf instillation, and the mean increase in NBD-associated fluorescence in ATII cells following Exosurf instillation. Our results suggest that sublethal hyperoxia decreases the short-term uptake but not the distribution of intratracheally instilled Exosurf.