Peroxisomes (PO) are a heterogeneous population of cell organelles which in mammals are most abundant in liver and kidney. Commonly, differential and density gradient centrifugation are used for their isolation which, however, give only rise to the so-called "heavy" PO with a buoyant density of 1.22-1.24 g/cm(3). Subpopulations other than the heavy PO which are also present in both of these tissues have escaped adequate purification because of their sedimentation characteristics which are close to those of other major organelles, in particular microsomes. Since the purification of these subpopulations has become an essential task in view of the putative importance of peroxisomal subpopulations in the biogenesis of this organelle, alternatives to density gradient centrifugation are required. Recently, we have introduced such a novel approach, named immune free flow electrophoresis (IFFE). IFFE combines the advantages of eletrophoretic separation with the high selectivity of an immune reaction. It makes use of the fact that the electrophoretic mobility of a subcellular particle, complexed with an antibody directed against the cytoplasmic domain of one of its integral membrane proteins is greatly diminished, provided the pH of the electrophoresis buffer is adjusted to pH approximately 8.0, the pI of immunoglobulin G (IgG) molecules. pH-values other than 8.0 proved to be less efficient, probably because IgG molecules only focus at pH 8.0 but are scattered at any other. Applying IFFE to heavy and light mitochondrial as well as microsomal fractions of rat liver not only regular PO (rho = 1.22-1.24 g/cm(3)) but also other subpopulations could be isolated. To substantiate the validity of this approach, we now have subfractionated mouse liver homogenates accordingly. Of the PO subpopulations collected, mainly that obtained from the heavy mitochondrial fraction differed in its composition of matrix and membrane proteins as revealed by immunoblotting. This is in line with the data reported on rat liver thus confirming the potential of IFFE in the isolation of distinct subpopulations of hepatic PO.