The proteoglycans and glycosaminoglycans of four human chondrosarcomas with different degrees of malignancy (I-III) have been studied. The hydrodynamic size of proteoglycan subunits and the tissue concentration of total glycosaminoglycans decreased with increasing grade of malignancy. The glycosaminoglycan distribution pattern of all chondrosarcomas showed a similar ratio of chondroitin-4-sulfate:chondroitin-6-sulfate but an increasing portion of keratan sulfate from grade I (6.5%) to grade III (19.2%). Determinations of the molecular weight (Mr values) of glycosaminoglycans were made after 3H labeling by alkaline reduction of proteoglycans in the presence of NaB3H4. The Mr of [3H]chondroitin sulfate isomers decreased markedly from grade I (35,500) to grade III (15,100) while the chain length of [3H]keratan sulfate showed minor variations (Mr 5600-6200). The previously reported decrease in the molecular weight of keratan sulfate with increasing degree of malignancy (S. Pal, W. Strider, R. Margolis, G. Gallo, S. Lee-Huang, and L. Rosenberg, 1978, J. Biol. Chem. 253, 1279-1289) was not observed.