Attenuated total reflection Fourier transform infrared spectroscopy was used to investigate the secondary structure of the surfactant protein SP-B. Nearly half of the polypeptide chain is folded in an alpha-helical conformation. No significant change of the secondary structure content was observed when the protein is associated to a lipid bilayer of dipalmitoylphosphatidylcholine (DPPC)/phosphatidylglycerol (PG) or of dipalmitoylphosphatidylglycerol (DPPG). The parameters related to the gamma w(CH2) vibration of the saturated acyl chains reveal no modification of the conformation or orientation of the lipids in the presence of SP-B. A model of orientation of the protein at the lipid/water interface is proposed. In this model, electrostatic interactions between charged residues of SP-B and polar headgroups of PG, and the presence of small hydrophobic alpha-helical peptide stretches slightly inside the bilayers, would maintain SP-B at the membrane surface.