Lipoprotein lipase (LPL) catalyzes the flux-generating step in transport of fatty acids from lipoprotein triacylglycerols into tissues for use in metabolic reactions. In vitro studies have shown that fatty acids can bind to the enzyme and impede its other interactions. In this study we have searched for evidence of fatty acid control of LPL in vivo by rapid infusion of a triacylglycerol emulsion to healthy volunteers. During infusion the activity of LPL but not of hepatic lipase increased in plasma, but to different degrees in different individuals. The time course for the increase in LPL activity differed from that for triacylglycerols but followed the plasma levels of free fatty acids. This was true during infusions and when the emulsion was given as a bolus injection. In particular there were several instances when plasma triacylglycerol levels were very high but free fatty acids and LPL activity remained low. Model studies with bovine LPL showed that fatty acids displace the enzyme from heparin-agarose. We suggest that in situations when fatty acids are generated more rapidly by LPL than they are used by the local tissue, they cause dissociation of the enzyme from its binding to endothelial heparin sulfate and are themselves released into circulation.