Pressure-area isotherms, Brewster angle microscopy, and grazing incidence X-ray diffraction measurements show that palmitic acid induces systematic changes in the physicochemical parameters and molecular organization of model lung surfactant lipid monolayers. Adding increasing fractions of palmitic acid (PA) to a 77/23 (wt/wt) mixture of dipalmitoylphosphatidylcholine (DPPC) and l-palmitoyl 2-oleyl-phosphatidyl-glycerol (POPG) increased the solid-phase fraction of the monolayer as shown by pressure-area isotherms and fluorescence and Brewster angle microscopy. Grazing incidence X-ray diffraction showed that the palmitic acid interacted specifically with the DPPC fraction to form a mixed crystalline solid phase with a tilt angle that decreased in Proportion to the fraction of palmitic acid. Increasing the PA content is roughly equivalent to increasing the surface pressure or decreasing the temperature of the DPPC/POPG monolayers. These results are important to the rational design of replacement lung surfactants for the treatment of respiratory distress syndrome.