A high-performance gel chromatography (HPGC) system has been developed which allows the unattended on-line determination of lipoprotein cholesterol distribution (VLDL-C, LDL-C, HDL-C), within 40 min, in microliter quantities of plasma using a single, relatively inexpensive column (Superose 6HR). The FAST cholesterol reagent (Sclavo) and a knitted PFTE Kratos reaction coil (Applied Biosystems) were found to provide optimal sensitivity, linearity, resolution, and dispersion characteristics. Validation is provided by comparison to target values for human quality control reference sera, and by comparing the values obtained by HPGC to the beta-quant method (LRC). The utility of the system is illustrated by comparing profiles from seven different species with normal or elevated plasma cholesterol concentrations. This technique allows rapid analysis of samples, regardless of species, without the use of precipitating agents or the ultracentrifuge. It could also be applied for the direct clinical determination of LDL-cholesterol.