An in vitro assay to study lipolysis of very low density lipoproteins (VLDL) by heparan sulfate proteoglycan (HSPG-bound lipoprotein lipase (LPL) was developed. Optimal conditions for VLDL lipolysis by HSPG-bound LPL were obtained by incubating plastic wells with 0.5 microg HSPG and 1.5 microg LPL, subsequently. Control experiments with heparinase indicate that at least 90% of the LPL activity is derived from LPL bound to heparan sulfate chains. For HSPG-LPL-mediated lipolysis, the apparent Km and Vmax values were 0.36 +/- 0.11 mM VLDL-triglycerides and 1.2 +/- 0.1 microM free fatty acids/min x ng LPL, respectively. The mean intra-assay and inter-assay coefficients of variance were 5% and 8%, respectively.