An improved system for electrophoresis of proteoglycans is presented, using a discontinuous buffer system that allows stacking of the sample. The molecular sieving in pure agarose was studied using cartilage proteoglycans separated into fractions of different size (K_av value) by gel chromatography. These fractions were used to establish the relationship between size and mobility in agarose gels of different concentrations. With low agarose concentration, the separation was largely dependent on charge density. With increasing agarose concentration, the separation became increasingly dependent on size. Electrophoresis provides a suitably sensitive tool for analysis of the proteoglycan and glycosaminoglycan fractions prepared by the Alcian blue precipitation described in the preceding paper. Alcian blue precipitated cartilage proteoglycans appeared identical in size to molecules prepared by conventional procedures when analyzed by electrophoresis. Proteoglycans of various sizes were prepared by precipitation with Alcian blue of 4 M guanidine-HCl extracts of bovine nasal cartilage, human articular cartilage, human skin, and bovine sclera. Proteoglycans were also precipitated from human synovial fluid and plasma. The samples were electrophoresed on 1.2% agarose gels, which was the highest agarose concentration suitable for large aggregating proteoglycans but still separating the smallest proteoglycans with one or two glycosaminoglycan chains from each other. Several sizes of proteoglycans were found in each sample except blood plasma, which only contained one small proteoglycan/glycosaminoglycan. Electrophoresis in agarose is also suitable for separating proteoglycan aggregates and monomers and allows rapid analysis of several samples at a time. The proteoglycans prepared by Alcian blue precipitation retained their ability to form aggregates with hyaluronic acid. In addition to separation according to size, the electrophoresis method is also useful for separating free glycosaminoglycan chains according to charge. Electrophoresis in 0.6% agarose was used to demonstrate that both chondroitin sulfate and keratan sulfate were precipitated with Alcian blue at 0.4 M guanidine-HCl. No keratan sulfate, either alone or together with chondroitin sulfate, was precipitated at 1.0 M guanidine-HCl concentration.