Background: We investigated the in vitro effects of various phospholipids as emulsifiers on the hydrolysing activities of lipoprotein lipase (LPL) Arg243His against triolein as substrate. LPL Arg243His, identified in a patient with hyperchylomicronaemia, displays severely diminished activity for triolein when emulsified with Triton X-100. Methods: Lipolytic activities of plasma obtained by heparin injection from a homozygous patient with LPL Arg243His were analysed using triolein emulsified with phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylserine (PS), phosphatidylinositol (PI), lysophosphatidylcholine (LPC), or Triton X-100 as substrates. Results: The hydrolysing activities of the patient’s plasma for triolein emulsified with PC, PE, PS, PI, LPC and Triton X-100 were 9.22 ± 1.06 μmol/ml/h/ngLPL, 2.94 ± 1.60 μmol/ml/h/ng LPL, 3.72 ± 1.63 μmol/ml/h/ng LPL, 3.40 ± 1.20 μmol/ml/h/ngLPL, 3.72 ± 1.96 μmol/ml/h/ngLPL and 7.80 ± 4.48 μmol/ml/h/ng LPL, respectively. Thus, the specific activities of the patient’s LPL determined with triolein emulsified with PC were significantly higher than those with PE, PS, PI or LPC as emulsifiers. Relative to the activities of normal plasma measured with PC, PE, PS, PI and LPC as emulsifiers, the mutant’s activities were 49.1 ± 5.2%, 44.1 ± 5.7%, 31.7 ± 12.6%, 19.2 ± 6.9% and 23.8 ± 11.3%, respectively. Using PC, PE, PS, PI and LPC as emulsifiers, the mutant’s activities for triolein-lipolysis relative to normal were significantly increased in comparison to the relative activity measured with the classical emulsifier, Triton X-100 (12.9 ± 6.7%). Conclusions: Impaired triolein hydrolysis by LPL Arg243His was partially ameliorated by triolein emulsification with phospholipids. The in vitro analysis of triolein hydrolysis using various phospholipid emulsifiers may be useful for the further understanding of impaired LPL function.