This report describes the densitometric quantitation of the two main HDL subfractions separated by electrophoresis in a polyacrylamide gradient gel, HDLS (mean apparent diameter 9.3 nm) and HDLL (mean apparent diameter 10.6 nm). The electrophoresis was carried out in a linear gradient of polyacrylamide ranging from 23 to 180 g/l on total sera prestained for lipid components by Sudan black B in ethylene glycol. HDL subfractions were quantified by scanning the gels at 633 nm with a laser densitometer. The precision was checked by intra-assay and between-assay studies (coefficient of variation 1.2 and 2.9%, respectively). The results were compared with the cholesterol content of HDL subfractions (r = 0.99) and with the HDL2:HDL3 distribution (r = 0.95). Reference values were obtained from a population of 214 normolipidemic subjects. They were in good agreement with those already published for HDL2 and HDL3. The method which needs only 4 microliter of serum, can be set up with currently available apparatus and is compatible with large series of samples, should allow large scale explorations of the variation of HDL subfraction distribution in normal and pathological populations.