A semi-automated method has been developed for determining alpha-lipoprotein cholesterol values. Precipitation of apolipoprotein B containing lipoproteins takes place in wells of microtiter plates after 100 microL of serum are mixed with 20 microL of a heparin/MnCl2 solution. A Beckman (Fullerton, CA) Biomek 1000 work station is used to transfer sera, supernatants and reagents between tubes and microtiter plates. Supernatant cholesterol is determined enzymatically, and absorbances are read at 490 nm using a Molecular Devices Corporation (Palo Alto, CA) plate reader. Values obtained on both fresh and frozen serum samples agreed with corresponding data obtained at the Centers for Disease Control (CDC; Atlanta, GA). For the fresh samples, the average bias was 2.87%. The within-run coefficients of variations were between 2.2 and 0.6% for the data obtained on CDC frozen control pools. The results indicate that the semi-automated method is suitable for obtaining accurate and precise data for alpha-lipoprotein cholesterol. The method lends itself to the analysis of large numbers of samples and is particularly suited for the study of lipoproteins of small mammals.