The structures of neutral and acidic glycosphingolipids from both normal colorectal epithelial cells and colorectal cancer cells, which were highly purified with the epithelial cell marker CD326, have been analyzed. The analysis was performed on samples from 16 patients. The carbohydrate moieties from glycosphingolipids were released by endoglycoceramidase II, labeled by pyridylamination, and identified using two-dimensional mapping and mass spectrometry. The structures from normal colorectal epithelial cells are characterized by dominant expression of neutral type-1 chain oligosaccharides. Three specific alterations were observed in malignant transformation; increased ratios of type-2 oligosaccharides, increased alpha2-3 and/or alpha2-6 sialylation and increased alpha1-2 fucosylation. Although the degree of alteration varies case to case, we found that two characteristic alterations tend to be associated with clinical features. One is a shift from type-1 dominant normal colorectal epithelial cells to type-2 dominant colorectal cancer cells. This shift was found in 5 patients having hepatic metastasis. The other is specific elevation of alpha2-3 sialylation observed in 2 cases exhibiting high serum levels of CA19-9. Examination of the activities of the related glycosyltransferases revealed that while some alterations could be accounted for by changes in the activities of related glycosyltransferases others could not. Although the number of cases analyzed is small, these findings provide valuable information which will help in the elucidation of the mechanism of synthesis of aberrant glycosylation and its involvement in cancer malignancy.