The miscibility and reactivity of binary monolayers of 1,2-dipalmitoyl-sn-glycero-3-phosphoglycolipoate (DPPGL) and 1-palmitoyl-2-(16-(S-methyldithio)hexadecanoyl)-sn-glycero-3-phosphocholine (DSDPPC) have been studied. The morphology of the binary monolayers transferred on a gold substrate at different surface pressures was determined by scanning probe microscopy (SPM) and compared with the interfacial properties of the films at the air/water interface. Specific immobilization of pepsin-cleaved antibody Fab' fragments from a Langmuir film onto a solid-supported DSDPPC-DPPGL (95:5 mol %) monolayer was utilized in the visualization of the miscibility and reactivity of the mixed lipid monolayers. The protein immobilization was shown to be dependent on the degree of condensation of the DSDPPC-DPPGL film. Furthermore, the linker molecules were shown to be favorably orientated for cross-linking reaction when amidst liquid-condensed domains of the matrix molecules. A closer look at the Fab' domains revealed, however, that the domains were not fully covered with antibodies. The lower than expected relative surface density (RSD) of the Fab' fragments was believed to be mainly due to random orientation of the Fab' fragments at the air-water interface, which prevented part of them from participating in a thiol-disulfide interchange reaction between the antibodies and the linker molecules.