BACKGROUND Plasma triglyceride analyses to evaluate the clearance of IV fat emulsion are necessary in patients receiving parenteral nutrition. Enzymatic kits for triglyceride analysis measure the glycerol that is hydrolyzed from triglyceride. This study investigates the effect of fat emulsion free glycerol on plasma triglyceride determination. METHODS Venous blood from fasting volunteers (n = 10) was drawn into tubes containing EDTA and was centrifuged to separate the plasma. An IV fat emulsion containing 2.25% glycerol was added to plasma samples to create serial dilutions ranging from 0.049% to 0.245% final lipid concentration. Total triglyceride and free glycerol concentrations were determined in each dilution and control sample. The free glycerol concentration was subtracted from the total triglyceride concentration to yield a true triglyceride value. RESULTS Increasing concentrations of fat emulsion added to donor plasma produced increases in total triglyceride and free glycerol concentrations. The increase in free glycerol concentration produced significant concentration-related differences between total and true triglyceride concentrations. CONCLUSIONS The total triglyceride level overestimated the true triglyceride concentration due to fat emulsion free glycerol. To assure reliable triglyceride results in patients receiving fat emulsion, blanking for free glycerol should be considered.