Hydrolysis of dissolved p-nitrophenyl acetate by pancreatic lipase follows the classical acyl enzyme pathway already proposed for other esterases. Kinetic parameters of the hydrolysis have been determined. The turnover rate of the reaction is many orders of magnitude slower than that for the natural emulsified substrates. Nevertheless, several arguments are in favor of the specificity of this hydrolysis: (1) triacetin, which resembles the usual substrates for the enzyme, is also hydrolyzed very slowly in solution; (2) dissolved triacetin and tripropionin are competitive inhibitors for the p-nitrophenyl acetate hydrolysis; (3) the same chemical structural features which are required in the case of emulsified substrates are also necessary to promote hydrolysis of dissolved p-nitrophenyl esters. This suggests that the same active site (or part of the same active site) is responsible for hydrolysis of both p-nitrophenyy acetate and specific emulsified substrates. Since deacylation is the rate-limiting step in the catalysis of p-nitrophenyl acetate, the intermediate acetyl enzyme can be isolated by trapping it at pH 5.0. Kinetic competence of this intermediate has been demonstrated. Hydrolysis by pancreatic lipase of dissolved monomeric p-nitrophenyl acetate and triacetin is considerably enhanced (100- to 500-fold) by various interfaces. This suggests that at least the deacylation step, which is rate limiting in absence of interface, is accelerated by the presence of inert interfaces. Siliconized glass beads were directly shown to accelerate the deacylation of isolated [3H]acetyl lipase by at least a hundred times. This step does not directly involve the ester substrate.Thus, it is suggested that a part of the activation of lipase at interfaces may be due to a conformational change resulting from adsorption.