Lipoprotein lipase (LPL) bound to endothelial cells is released from the cell surface by triacylglycerol-rich lipoproteins and oleic acid (Saxena, U., Witte, L.D. and Goldberg, I.J. (1989) J. Biol. Chem. 264, 4349-4355). Studies were conducted to compare the ability of different fatty acids to release 125I-labelled bovine milk LPL bound to endothelial cells and to define the mechanism for this effect. Using fatty acid/bovine serum albumin (BSA) solutions (molar ratio, 6:1), the release of LPL from endothelial cell surfaces using monounsaturated (18:1), polyunsaturated (18:2) and saturated (16:0) fatty acids was 78%, 60% and 28%, respectively. Release of LPL from heparin-agarose followed a similar pattern, suggesting that the fatty acids specifically affected LPL-heparin interaction. Short-chain fatty acids (C6, C8 and C10), medium-chain fatty acids (C12 and C14) and elaidic acid, a transisomer of oleic acid, released less 125I-LPL than oleic acid from heparin-agarose. To determine whether oleic acid release of 125I-LPL from heparin-agarose was due to binding of the fatty acid to heparin or LPL, oleic acid was incubated with either LPL or heparin-agarose prior to performing the affinity chromatography. Only the prior incubation with LPL affected the binding to heparin-agarose. This demonstrates that dissociation of LPL from heparin required interaction of fatty acid with LPL. At high molar ratios of fatty acid:BSA (greater than 3:1), apo C-II is known to be ineffective as an activator of LPL. To determine whether this effect is due to decreased association of apo C-II with LPL, 125I-apo C-II (0.5-10 nmol) was allowed to bind to LPL-Sepharose. A 6:1 molar ratio of oleic acid:BSA produced up to 69% decrease in the amount of 125I-apo C-II bound to the gel. This dissociation of apo C-II from LPL by oleic acid was also demonstrated using gel-filtration chromatography. Thus, the amount and type of fatty acids may be important in regulating LPL activity in vivo by affecting both LPL interaction with glycosaminoglycans and with apo C-II.