In a double-labeling experiment to detect any change in the composition of glycoproteins and glycolipids following oncogenic virus transformation of mouse fibroblasts, [3H]- and [14C]glucosamine were used to label cell line 3T3 and SV-40 virus-transformed 3T3 cells. After growing separately in the presence of radioactive glucosamine, 3T3 and SV-40-3T3 cells were harvested and mixed in the following combinations: [3H]SV-40-3T3 and [14C]3T3 (mixture I), and [3H]3T3 and [14C]SV-40-3T3 (mixture II). Both mixtures were homogenized and separated into subcellular fractions by the method of Wallach. The 3H to 14C ratios were also determined for sialic acid, galactosamine, and glucosamine isolated from each fraction. Analysis of the relative composition of membrane glycoproteins and glycolipids in 3T3 and SV- 40-3T3 showed a much lower sialic acid and galactosamine content and a reciprocal increase in the relative content of glucosamine in SV-40-3T3 as compared with 3T3. These differences in the relative composition of amino sugars between 3T3 and SV-40-3T3 were seen in all particulate fractions but not in the nucleotide sugar fraction. Thus the changes in the membrane carbohydrate composition in virus-transformed cells could not be attributed to a lack of particular nucleotide sugar precursors. Measurements of the absolute amounts of neutral and amino sugars present in the particulate fractions of 3T3 and SV-40-3T3 by the isotope dilution technique of Ginsburg revealed a marked decrease in both neutral and amino sugars per milligram of protein in SV-40-3T3 as compared with 3T3. Although the double-labeling technique had revealed minor differences in the relative composition of membrane carbohydrates between 3T3 and spontaneously transformed 3T3, measurements of sugar contents in spontaneously transformed 3T3 by the isotope dilution technique nevertheless showed decreased amounts of both neutral and amino sugars with the levels of sugar per milligram of protein intermediate between those present in 3T3 and SV-40-3T3. The implications of these changes in membrane glycoproteins and glycolipids following viral and spontaneous transformation are discussed. © 1969, American Chemical Society. All rights reserved.