Clathrin-coated vesicles have been isolated from bovine brain. To allow their further use in biophysical studies, the homogeneity of the preparations has been fully characterized after chromatography on Sephacryl S-1000, which is employed in many studies. It is demonstrated here that clathrin-coated vesicles are not stable on the gels and that their instability is increased in preparations using gels that are not presaturated with phospholipids. In addition, some fractionation occurs during chromatography. It is proposed that the slower migrating fractions contain mainly empty clathrin coats. Changes that occurred during the chromatography step are the result of reversible and irreversible events and are probably related to the assembly/disassembly cycle of clathrin observed in vitro.