AbstractFluorescent esters containing pyrenedecanoic acid (P10) or pyrenebutanoic (P4) acid (P4cholesterol, P10cholesterol, P4‐ and P10‐containing triacylglycerols) were synthesized and used as substrates for human pancreatic carboxylic ester hydrolase and bile salt‐stimulated lipase from human milk. Both enzymes were purified by immunoaffinity chromatography. All fluorescent pyrene derivatives were hydrolyzed by pancreatic carboxylic ester hydrolase and bile salt‐stimulated lipase, but at different rates. The hydrolytic rates of the “short” acyl esters (P4‐containing esters) were higher than those of the “long” ones (P10‐containing esters). Conditions were optimized for sensitivity of the assay using fluorescent cholesteryl esters. The pH optimum was 7.5–8.0. Sodium cholate exhibited a stronger activating effect than taurocholate or taurodeoxycholate (maximal activation was achieved with 5 mmol/L cholate and with a molar ratio cholesteryl ester/cholate around 1∶10). Both pancreatic carboxylic ester hydrolase and bile salt‐stimulated lipase from milk were strongly inhibited by the other amphiphiles tested, namely phosphatidylcholine and Triton X‐100, and were inactivated by low concentrations (10 μmol/L) of the serine‐reactive diethyl‐paranitrophenyl phosphate (E600). Both enzymes were strongly inhibited by relatively low concentrations of plasma low density lipoproteins. These studies indicate that the fluorescent esters containing pyrene fatty acids can be used as substrates for assaying and investigating the properties of pancreatic carboxylic ester hydrolase as well as bile salt‐stimulated lipase from milk.