Insertion properties of the antibiotics Lasalocid and Monensin sodium salts (LAS-Na and MON-Na) in Langmuir monolayers of two different lipid molecules, dipalmitoylphosphatidylcholine (DPPC) and octadecanol (C180H), have been investigated. Results from compression isotherms of the films on pure water and on a biologically buffered subphase have been compared. Values of the molecular areas of mixed films of antibiotic/lipid and of the molecular areas of the pure lipids, at a given surface pressure, have been used to calculate the aggregation numberkof the solute molecules in a concentration range of ρ (moles of antibiotic/moles of lipid) from 0.005 to 0.1. Theknumber in the film of DPPC increases when ρ increases, it is enhanced on the buffered subphase compared to the subphase of water and it is more important for MON-Na than for LAS-Na. There is hardly any evidence ofaggregation in the films of C180H. The two lipid systems both have in common that the mixed films with LAS-Na on the buffered subphase are the most pressure resistant and the ones from which the antibiotic is the least easily expelled.