The four-component system acyl chain perdeuterated 1,2-dipalmitoylphosphatidylcholine (DPPC)/1,2-dipalmitoylphosphatidylglycerol/ (DPPG)/pulmonary surfactant protein SP-C/cholesterol provides a useful model for in vitro biophysical studies of the reversible monolayer to multilayer transition that occurs during compression <-> expansion cycles in the lung. Monolayer films of this mixture (with chain perdeuterated DPPC-d(62)) at the air/water interface have been transferred to solid substrates under conditions of continuously varying surface pressure, an approach termed COVASP (continuously varying surface pressures) (Langmuir 2007, 23, 4958). The thermodynamic properties of the Langmuir films have been examined with pressure-area isotherms, while the molecular properties of the film constituents in the transferred films in the monolayer and multilayer phases have been examined with IR spectroscopy. Quantitative intensity measurements of the DPPC-d(62), DPPG, and SP-C components in each phase reveal that the DPPG and SP-C constituents are relatively enriched in the multilayer compared with the DPPC-d62, although all three species are present in both phases. Some molecular structure information is available from the surface-pressure-induced variation in IR parameters. The DPPC-d(62) exhibits slightly increased conformational order in the multilayer phase as detected from decreases in the CD2 stretching frequencies upon compression, while the lipid phosphate residues become dehydrated, as deduced from increases in the 1245 cm(-1) symmetric PO2- stretching frequency. A small increase is observed in the protein amide I frequency; possible interpretations of these changes are presented. The current observations are compared with ideas contained in the "squeeze-out hypothesis" (Handbook of Physiology, The Respiratory System; American Physiological Society Press: Bethesda, MD, 1986; Vol. 111, p 247) and in the "liquid crystalline collapse" model (Biophys. J. 2003, 84, 3792). Within the limitation of the current procedures, the data contain elements from both these descriptions of the monolayer transformation. Extensions and possible limitations of the COVASP-IR method are discussed.