Fluorescence microscopy, surface potential, and activity measurements were used to investigate the influence of fatty acids and fatty alcohols on the lipolytic activity of several lipases. We have determined the lateral lipid distribution and interfacial properties of Langmuir mixed monolayers composed of 1,2-didecanoylglycerol/eicosanoic acid or 1,2-didecanoylglycerol/1-octadecanol molecules and have measured lipase activities toward these films. Enzymatic activities are remarkably influenced by the addition of fatty acid. Activity decreases continuously up to a mole fraction of ≈ 0.1 fatty acid, where phase separation and a change in surface potential are observed. Higher concentrations of fatty acid have only marginal effects on the lipase activities. The relative activity between the different lipases varies substantially, and there is an indication that the level of inhibition correlates with the isoelectric point (pI) of the enzymes. A simpler mechanism is observed by the addition of fatty alcohol. Within the concentration range studied, 1-octadecanol is immiscible in the diacylglyceride matrix, forming liquid-condensed domains. The inhibitory effect is related to the reduction of available diacylglyceride area to the enzyme. Direct imaging of the lipolytic hydrolysis of these monolayers show that relatively small domains are formed, suggesting that the enzyme preferentially acts on pure diacylglyceride patches.