We have developed a high resolution microvolume Vertical Auto Profile (VAP) method for the simultaneous measurement of cholesterol in all lipoprotein classes, including lipoprotein[a] (Lp[a]) and intermediate density lipoprotein (IDL). This method, designated as VAP-II, uses a nonsegmented continuous flow (controlled-dispersion flow) analyzer for the enzymatic analysis of cholesterol in lipoprotein classes separated by a short spin (47 min) single vertical ultracentrifugation. Cholesterol concentrations of high (HDL), low (LDL), very low (VLDL), and intermediate (IDL) density lipoproteins, as well as Lp[a], are determined by decomposing the spectrophotometric absorbance curve, obtained from the continuous analysis of the centrifuged sample, into its components using software developed in this laboratory. Analysis by VAP-II is rapid and sensitive (as little as 40 μl plasma is required per assay). The resolution of lipoprotein peaks is considerably enhanced in the present analyzer compared to the previous analyzer (VAP-I, which used the Technicon AutoAnalyzer); improvement is especially noticeable for Lp[a] and IDL. Total and lipoprotein cholesterol values obtained by VAP-II correlated well with the values obtained by Northwest Lipid Research Laboratories (NWLRL). VAP-II Lp[a] cholesterol values also correlated well with the Lp[a] mass values obtained by an immunoassay technique performed at NWLRL (r = 0.907). The reproducibility and accuracy of the method are within the requirements of the CDC-NHLBI (Centers for Disease Control-National Heart, Lung, and Blood Institute) Lipid Standardization Program.