AbstractA fluorescent radiolabeled triacylglycerol has been synthesized by using a fluorescent fatty acid (pyrene decanoic acid) and a radiolabeled oleic acid. This analog of the natural substrate, 1(3)pyrene decanoic‐2,3(1,2)‐dioleoyl‐sn‐glycerol, has been tested as substrate for determining lipoprotein lipase and hepatic triacylglycerol lipase activities in post‐heparin plasma. Optimal conditions for the determination of the two post‐heparin plasma lipases were similar to those using radiolabeled triolein. Using this substrate, both post‐heparin lipases exhibited their characteristic properties (pH optimum and effect of inhibitors) and attacked external ester bonds (1 or 3) containing pyrene decanoic and oleic acids at a similar rate