Rationally designed peptide biosurfactant AM1 was mixed with sodium dodecyl benzene sulfonate (SDOBS) to self-assemble a mixed surfactant-biosurfactant layer at the air-water interface. Under optimal conditions in the presence of Zn2+, the interfacial elasticity of the mixed layer was approximately 5-fold higher than for biosurfactant alone. Two head positional isomers, SDOBS-2 and SDOBS-6, were compared for their ability to enhance interfacial film strength. SDOBS-6 forms a stronger layer with AM1 than does SDOBS-2. The highest interfacial elasticity of the AM1/SDOBS-6 layer was 640 mN m(-1) whereas the maximum value for the AM1/SDOBS-2 layer was 440 mN m(-1). Neutron reflection was used to investigate the structure of AM1/SDOBS films at varied bulk SDOBS concentrations. Both deuterated and nondeuterated SDOBS-2 and SDOBS-6 were used to provide contrast variation. It was shown that there is cooperative interaction between AM1 and SDOBS at low SDOBS concentration in the presence of 100 microM Zn2+, promoting AM1 adsorption atthe interface to form a two-layered structure of AM1 resulting in a mechanically strong interfacial film. In the presence of EDTA, only a single AM1 layer was formed at the same SDOBS concentration, and the film did not show lateral force transmission capability. Further increasing the SDOBS concentration to a molar excess of > 10x decreased the peptide population at the interface and resulted in a mechanically weak layer. Compared to SDOBS-6, SDOBS-2 depletes AM1 at a lower bulk concentration. These results demonstrate that the film strength of a self-assembled surfactant-biosurfactant mixed layer can be fine tuned by changing the isomer type and concentration of surfactant and by adding or removing metal ions.