Surfactant obtained from bronchoalveolar lavage (BAL) can be separated into subfractions based on sedimentation characteristics. It has been suggested that the 10,000 x g, 60,000 x g and 100,000 x g subfractions isolated by this approach represent stages of surfactant extracellular processing. These three subfractions have been reported to differ in their morphology, composition and ability to lower surface tension. We wished to determine if infrared spectroscopy, which may be applied as a non-invasive technique could potentially prove useful for characterization and quantification of bronchoalveolar lavage (BAL) protein and phospholipid, and if this approach could detect differences in intermediate surfactant processing stages. Subfractions were collected from adult rabbit lungs by BAL and differential centrifugation and analyzed by Fourier transform infrared (FT-IR) spectroscopy. Biochemical assay of phospholipid and protein showed differences between subfractions that correlated well with the phospholipid/protein ratios obtained from FT-IR spectra (r = 0.939; r2 = 0.882). The subfraction sedimenting at 100,000 x g (P100) exhibited spectral shifts in the Amide I band, suggesting that the protein secondary structure was different compared to other fractions. Spectra obtained after separation of lipids and protein components showed an apparent disordering of protein secondary structure but little or no effect on the structure or mobility of phospholipids. These results support the idea that subfractions represent various processing stages of surfactant. In addition, they show that results from FT-IR analyses correlate significantly with traditional biochemical assay methods which may prove of clinical use.(ABSTRACT TRUNCATED AT 250 WORDS)