The present work evaluates the kinetics of the interaction of S-layer protein from Lactobacillus brevis with lipid monolayers by measuring the changes in the surface pressure as a function of time for different lipid compositions and at different lateral compressions. At high surface pressures, or at high cholesterol ratios, in which membrane rigidity and surface polarity are increased, the kinetics can be described by a pure diffusional process. At low pressures or in the absence of cholesterol, the kinetics of protein interaction can be interpreted as a consequence of a relaxation process of the membrane structure coupled to diffusion. As the less packed monolayers are more hydrated, the relaxation processes at low initial surface pressures could be ascribed to changes in water organization in the membrane. These observations denote that kinetic insertion of proteins can be modulated by components that modify the hydration state of the interface.