When the glycosaminoglycans (GAGs) of BALB/c 3T3 and B16.F10 melanoma cells were compared during parallel studies, substantial differences were found in the cellular content, rates of turnover, and extent of intrinsic sulfated GAG degradation between the two cell types. The 3T3 cells had more than 10-fold more metabolically-labeled [35S]-GAGs per cell under steady state conditions than did the melanoma cells, and in pulse-chase studies they turned this material over at a much slower rate (apparent respective half-times of 30 hours and 8 hours). Moreover, the labeled material released into the medium during the chase periods was approximately 43% low molecular weight polymeric product, eluting in the included volume of Sephadex G50, for the 3T3 cells, as compared with 79% for the B16.F10 line. For both cell lines approximately 86% of total cell layer-associated polymeric GAG was accessible to GAG-degrading enzymes under conditions which did not lyse the cells, indicating that the majority of cellular GAGs is present external to the plasma membrane. The extensive digestion of sulfated GAGs in evidence for the melanoma cells suggests the involvement of constitutively high cellular levels of GAG-degrading enzymes.