Penicillium cyclopium produces two lipases with different substrate specificities. Lipase I is predominantly active on triacylglycerols whereas lipase II hydrolyzes mono- and diacylglycerols but not triacylglycerols. In this study, we compared the kinetic properties of P. cyclopium lipases and human pancreatic lipase, a classical triacylglycerol lipase, by using vinyl esters as substrates. Results indicate that P. cyclopium lipases I and II and human pancreatic lipase hydrolyze solutions of vinyl propionate or vinyl butyrate at high relative rates compared with emulsions of the same esters, although, in all cases, maximal activity is reached in the presence of emulsified particles, at substrate concentrations above the solubility limit. It appears that partially water-soluble short-chain vinyl esters are suitable substrates for comparing the activity of lipolytic enzymes of different origin and specificity toward esters in solution and in emulsion.