Epifluorescence microscopy was used to study the structure and phase behavior of phospholipid films containing a human-sequence monomeric SP-B 1-25 synthetic peptide (mSP-B 1-25). Measurements were done directly at the air-water (A/W) interface on films in a Langmuir-Whilhelmy balance coupled to a fluorescence microscope and real-time detection system to yield an approximate optical resolution of 1 μm. Fluorescence was achieved by laser excitation of 2-(4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene- 3-dodecanoyl)-1-hexadecanoyl-sn-glycero-3-PC (BODIPY-PC, concentration ≤1 mol%). The presence of mSP-B 1-25 in films of 4:1 (mol/mol) 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC)/1,2-dioleoyl-sn-glycero-3- [phospho-rac-(1-glycerol)] (sodium salt) (DOPG) had a substantial effect on lipid morphology and phase behavior that depended on both surface pressure and peptide concentration (10, 5, and 1 wt.%). The mSP-B 1-25 peptide tended to fluidize phospholipid monolayers based on expanded molecular areas and reduced collapse pressures. In addition, epifluorescence measurements revealed the formation of solid-phase domains apparent as three-armed counterclockwise spirals separated from regions of fluid liquid-expanded phase domains in compressed phospholipid-peptide films. The appearance of these separated solid-phase domains resembled pure L-DPPC rather than the ensemble-type solid domains found in films of DPPC/DOPG alone and were most apparent when 10 wt.% mSP-B 1-25 was present. In contrast, films containing lower, more physiological mSP-B 1-25 contents of 5 and 1 wt.% exhibited a prominent intermediate 'dendritic' phase that increased in extent as surface pressure was raised. This phase was characterized by branching structures that formed a lattice-like mesh network with fluorescence intensities between a dye-depleted solid domain and a dye-enriched liquid phase. These results indicate that mSP-B 1-25 at near-physiological levels produces morphological changes in phospholipid monolayers analogous to those observed for native SP-B 1-79. © 2004 Elsevier B.V. All rigths reserved.