Two-dimensional phase separation in alpha(s)-casein/beta-casein/water ternary film adsorbed at the air-water interface has been studied using an epi-fluorescence microscopy technique. The equilibrium composition of saturated mixed monolayer films of alpha(s)-casein/beta-casein at the air-water interface at various bulk concentration ratios did not follow a Langmuir-type competitive adsorption model. This was due to a change in the binding affinity of the proteins to the interface as a result of incompatibility of mixing of the proteins in the mixed monolayer. This manifested itself in two-dimensional phase separation of alpha(s)-casein and beta-casein in the mixed protein film. alpha(s)-Casein always tended to be the dispersed phase and beta-casein the continuous phase in the film. Also, phase separation occurred only when the film at the air-water interface was aged for several days, suggesting that the process was kinetically limited by lateral diffusion of the protein components in the viscous film.