Serum high density lipoprotein (HDL) cholesterol was measured in non-diabetic and diabetic men and women by ultracentrifugation, heparin-manganese chloride precipitation (heparin/Mn++), and sodium phosphotungstate-magnesium chloride precipitation (PT/Mg++). Results of the three methods were closely correlated in both diabetics and non-diabetics. Concentrations were, however, generally lowest by PT/Mg++, intermediate by ultracentrifugation, and highest by heparin/Mn++. These differences were small magnitude except in the case of diabetic patients treated with insulin. In these patients, serum HDL cholesterol levels were significantly greater than those of non-diabetic patients when measured by heparin/Mn++ and ultracentrifugation, but not by PT/Mg++. This was explained by the finding that the discrepancy between PT/Mg++ and ultracentrifugation increased with increasing HDL cholesterol concentration.