1,2-Dimethylhydrazine is a procarcinogen with selectivity for the colon. In weekly s.c. doses of 20 mg/kg of body weight, this agent produces colonic tumors in virtually 100% of rodents, with a latency period of approximately 6 months. To determine whether alterations in the glycosphingolipid content and composition of rat colonic epithelial cells existed before the development of dimethylhydrazine-induced colon cancer, rats were given s.c. injections of this agent (20 mg/kg body weight per week) or diluent for 5 weeks. Animals were sacrificed at this time period and colonocytes isolated from each group. Glycosphingolipids were then extracted from these cells and analyzed by thin-layer chromatography, high-performance liquid chromatography and gas-liquid chromatography. The results of these studies demonstrate that: (a) the content and relative percentages of globotriaosylceramide is increased, whereas hematoside and globotetraosylceramide are decreased in dimethylhydrazine-treated colonocytes compared to their control counterparts; and (b) differences in the enzymatic activities responsible for the biosynthesis of these glycosphingolipids appear to explain, at least partially, these compositional differences. The present data, therefore, suggest that alterations in certain glycosphingolipids may be an early event in colonic malignant transformation and, furthermore, that these alterations may prove useful in the detection of early colon cancer.