The location and depth of each residue of lung pulmonary surfactant protein B (SP-B(1-25)) in a phospholipid bilayer (PB) was determined by fluorescence quenching using synthesized single-residue-substituted peptides that were reconstituted into 1,2-dipalmitoyl phosphatidylcholine (DPPC)-enriched liposomes. The single-residue substitutions in peptides were either aspartate or tryptophan. The aspartate was subsequently labeled with the N-cyclohexyl-N'-(4-(dimethylamino)naphthyl)carbodiimide (NCD-4) fluorophore, whereas tryptophan is autofluorescent. Spin-labeled compounds, 5-doxylstearic acid (5-DSA), 7-doxylstearic acid (7-DSA), 12-doxylstearic acid (12-DSA), 4-(N,N-dimethyl-N-hexadecyl)ammonium-2,2,6,6-tetramethylpiperidine-1-oxyl iodide (CAT-16), and 4-trimethylammonium-2,2,6,6-tetramethylpiperidine-1-oxy iodide (CAT-1), were used in the quenching experiments. The effective quenching order is determined by the accessibility of the quencher to a fluorescent group on the peptide. The order of quenching efficiency provides information about the relative locations of individual residues in the PB. Our data indicate that residues Phe1-Pro6 are located at the surface of PB, residues Tyr7-Trp9 are embedded in PB, and residues Leu10-Ile22 are involved in an amphipathic alpha-helix with its axis parallel to the surface of PB; residues Pro23-Gly25 reside at the surface. The effects of intermolecular disulfide bond formation in the SP-B(1-25) dimer were also investigated. The experiments suggest that the SP-B helix A has to rotate at an angle to form a disulfide bond with the neighboring cysteine, which makes the hydrophobic sides of the amphipathic helices face each other, thus forming a hydrophobic domain. The detailed topographical mapping of SP-B(1-25) and its dimer in PB provides new insights into the conformational organization of the lung pulmonary surfactant proteins in the environment that mimics the native state. The environment-specific conformational flexibility of the hydrophobic domain created by SP-B folding may explain the key functional properties of SP-B including their impact on phospholipid transport between the lipid phases and in modulating the cell inflammatory response during respiratory distress syndrome.