Interactions between different amphiphiles and Rhizomucor miehei lipase were investigated by a variety of techniques. Complex formation in aqueous bulk solution was studied using surface tension measurements. Interactions at the oil-water and the solid-water interfaces were investigated by measuring mobility of emulsion droplets and by ellipsometry, respectively. The results from the different methods were coherent and indicated that cationic surfactants form complex with the lipase over a broad pH range, also below the isoelectric point of the lipase. No such interaction were found for neither anionic or nonionic surfactants. It is postulated that the interaction between cationic surfactants and lipase is due to a combination of electrostatic attraction and hydrohobic interaction and that no such combined interaction occurs with anionic surfactants. The interaction between cationic surfactant and lipase leads to a reduction of reaction rate in lipase-catalyzed hydrolysis of a palm oil. It is also shown that in the same model reaction a normal straight chain alcohol ethoxylate is a substrate for the lipase. An appreciable amount of fatty acid ester of the surfactant is formed as biproduct of the reaction. Branched-tail alcohol ethoxylates are not substrates and appear not to be competitive inhibitors for the enzyme. Likewise, the double-tailed ester surfactant sodium bis(2-ethylexyl)sulfosuccinate (AOT) seems not to interact with the enzyme active site. Thus, anionic and nonionic surfactants with bulky hydrophobic tails are the preferred surfactants for microemulsion-based reactions with Rhizomucor miehei lipase as catalyst.