The hydrolysis of long-chain alkanoylcholines catalyzed by butyrylcholinesterase (EC 3.1.1.8) has been studied. Radiolabelled substrates have been used and a radiochromatographic detection method developed earlier has been applied. The long-chain choline esters were found to be excellent substrates for butyrylcholinesterase at low concentrations, with Km values lower than those of short-chain analogues. At higher substrate concentrations, however, the hydrolysis reaction is inhibited, due to the formation of mixed micelles between the amphiphilic substrate and the corresponding alkanoic acid formed in the hydrolysis reaction. The inhibition may also partially be the result of conformational changes of the protein following adsorption of the cationic amphiphile. Critical micelle concentrations (CMC) for the long-chain substrates, as well as for mixed micelles, have been determined.