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 μg HSPG and 1.5 μg 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 K(m) and V(max) values were 0.36 ± 0.11 mM VLDL-triglycerides and 1.2 ± 0.1 μM free fatty acids/min·ng LPL, respectively. The mean intra-assay and inter-assay coefficients of variance were 5% and 8%, respectively.