The conformation and orientation of synthetic monomeric human sequence SP-B 1-25 (mSP-B 1-25) was studied in films with phospholipids at the air-water (A/W) interface by polarization modulation infrared reflectance absorption spectroscopy (PM-IRRAS). Modified two-dimensional infrared (2D IR) correlation analysis was applied to PM-IRRAS spectra to define changes in the secondary structure and rates of reorientation of mSP-B 1-25 in the monolayer during compression. PM-IRRAS spectra and 2D IR correlation analysis showed that, in pure films, mSP-B 1-25 had a major α-helical conformation plus regions of β-sheet structure. These α-helical regions reoriented later during film compression than β structural regions, and became oriented normal to the A/W interface as surface pressure increased. In mixed films with 4:1 mol:mol acyl chain perdeuterated 1,2-dipalmitoyl-sn-glycero-3-phosphocholine/1,2-dioleoyl-sn- glycero-3-[phospho-rac-(1-glycerol)] (sodium salt) (DPPC-d 62:DOPG), the IR spectra of mSP-B 1-25 showed that a significant, concentration-dependent conformational change occurred when mSP-B 1-25 was incorporated into a DPPC-d 62:DOPG monolayer. At an mSP-B 1-25 concentration of 10 wt.%, the peptide assumed a predominately β-sheet conformation with no contribution from α-helical structures. At lower, more physiological peptide concentrations, 2D IR correlation analysis showed that the propensity of mSP-B 1-25 to form α-helical structures was increased. In phospholipid films containing 5 wt.% mSP-B 1-25, a substantial α-helical peptide structural component was observed, but regions of α and β structure reoriented together rather than independently during compression. In films containing 1 wt.% mSP-B 1-25, peptide conformation was predominantly α-helical and the helical regions reoriented later during compression than the remaining β structural components. The increased α-helical structure of mSP-B 1-25 demonstrated here by PM-IRRAS and 2D IR correlation analysis in monolayers of 4:1 DPPC:DOPG containing 1 wt.% (and, to a lesser extent, 5 wt.%) peptide may be relevant for the formation of the intermediate order 'dendritic' surface phase observed in similar surface films by epi-fluorescence. © 2004 Elsevier B.V. All rights reserved.