In situ external reflection infrared spectroscopy at the air-water interface was used to study the influence on phospholipid structure of an endogenous mixture of the two hydrophobic surfactant proteins, SP-B and SP-C, which are thought to play pivotal roles in the adsorption and function of pulmonary surfactant. Mixtures studied were 1:1, 2:1, and 7:1 (mol:mol) DPPC-d62:DPPG, and 7:1 DPPC-d62:DOPG, alone and in the presence of 0.5-10 wt % mixed SP-B/C purified chromatographically from calf lung surfactant extract. Perdeuteration of DPPC produced a shift in vibrational frequencies so that it could be differentiated spectroscopically from the phosphoglycerol component in the surface monolayer. CH2 antisymmetric and symmetric stretching bands (∼2920 and 2852 cm-1) along with the analogous CD2 stretching bands (∼2194 and 2089 cm-1) were analyzed, and band heights and peak wavenumber positions were assessed as a function of monolayer surface pressure. Small, near-physiological contents of 1-2 wt % SP-B/C typically produced the maximum observed spectroscopic effects, which were abolished at high protein contents of 10 wt %. Analysis of CH2 and CD2 stretching bands and C-H/C-D band height ratios indicated that SP-B/C affected PC and PG lipids differently within the surface monolayer. SP-B/C had preferential interactions with DPPG in 1:1, 2:1, and 7:1 DPPC-d62:DPPG films that increased its acyl chain order. SP-B/C also interacted specifically with DOPG in 7:1 DPPC-d62:DOPG monolayers, but in this case an increase in CH2 band heights and peak wavenumber positions indicated a further disordering of the already fluid DOPG acyl chains. CD2 band height and peak wavenumber analysis indicated that SP-B/C had no significant effect on the structure of DPPC-d62 chains in 7:1 films with DPPG or DOPG, and had only a slight tendency to increase the acyl chain order in 1:1 films of DPPC-d62:DPPG. SP-B/C had no significant effect on DPPC-d62 structure in films with DOPG. Infrared results also indicated that interactions involving SP-B/C and lipids led to exclusion of PC and PG lipids from the compressed interfacial monolayer, in agreement with our previous report on the phase morphology of lipid monolayers containing 1 wt % SP-B/C.