The breakdown of normal substrates by lipases requires an interfacial binding step prior to hydrolysis. Interfacial binding and subsequent hydrolysis will be affected by the lipid components and hence physical properties of the substrate surface. In order to investigate in detail the effect of lipid structure on the activity of lipoprotein lipase (LPL), triolein-containing emulsion particles of defined composition have been used as substrates. In addition, lipase activity has been measured using a continuous fluorescence displacement assay that monitors the release of long-chain fatty acids as an alternative to normal radiochemical assays. Using this fluorescence assay, rates of hydrolysis of triolein were the same as when using a standard radiochemical assay under identical conditions. Activation by apolipoprotein CII was very similar by both methods; however, the extent of activation (2-3-fold) was less than has been reported previously using different assay conditions. In order to investigate the effect of cholesterol on LPL activity, emulsion particles were prepared in which the cholesterol/egg-phosphatidylcholine ratio was increased up to a 1:1 molar ratio. A pronounced stimulatory effect of cholesterol was observed under these assay conditions, with up to a 5-fold increase in rate compared with emulsion particles without cholesterol. Since high molar ratios of cholesterol are reported to exclude triacylglycerol from the phospholipid surface [Spooner and Small (1987) Biochemistry 26, 5820-5825], these results are not consistent with a mechanism involving LPL hydrolysis of surface triacylglycerol. Instead, they support an interfacial penetration model, allowing the enzyme's active site direct access to triacylglycerol in the lipoprotein core. Perturbation of the surface phospholipid monolayer of the emulsion particle as a result of hydrolysis by Naja naja phospholipase A2 resulted in a 10-fold activation of LPL, providing further support for an interfacial penetration model. The stimulatory effect of apolipoprotein CII was not modulated by modification of the interface with cholesterol.