Diabetic and normal cell lines from Chinese hamster kidneys were cultured in media containing 35SO4 and 3H-glucosamine. Glycosaminoglycans (GAG) were extracted and analyzed from the media, trypsin, and cell pellet by enzymatic and electrophoretic procedures. Significant increases in the hyaluronic acid content were noted in all three fractions of diabetic GAGs when compared with normals. In addition, an increased heparan sulfate content and decreased chondroitin sulfate amounts were noted in diabetic cell lines. These data suggest that in vivo changes in GAG types and amounts in diabetic kidneys seen by others may also be seen in cultured cells.