The soy isoflavone genistein can affect cell metabolism by specifically inhibiting protein tyrosine kinase (PTK) and/or interacting with the estrogen receptors (ERs). Glycosaminoglycans (GAG)/proteoglycans (PG) may participate in tumor development and progression. The synthesis of GAG by two human colon cancer cell lines, HT-29 and SW-1116, and the effects of genistein on their production and distribution between culture medium and cell membrane were studied. The mitogenic activity of genistein on both cell lines growth was also examined. Metabolic labeling, sensitive high pressure liquid chromatography (HPLC) techniques and fluorometric cell proliferation assays were utilized. The results demonstrate that both estrogen receptor beta-positive (ERbeta+) cancer cell lines produced hyaluronan (HA), both extracellular and membrane-associated galactosaminoglycans (GalAG) and heparan sulfate (HS), with the HT-29 cells producing all GAG fractions at significantly higher rates. The observed dose-dependent inhibitory effect of genistein on the synthesis of both secreted and cell-associated GAG/PG by the SW-1116 cells, as well as on their growth, was suggestive of a PTK mechanism. On the other hand, the synthesis of GAGs/PGs by HT-29 cells in the presence of genistein was dependent on their type and localization which implies the active participation of the ERs, which was further supported by the observed growth stimulation at low concentrations of genistein.