D-Galactosamine reduces the hepatic content of uridine phosphates, UDP-galactose, and UDPglucose due to an accumulation of UDP-amino sugars; this deficiency can result in severe hepatocellular damage. Alterations of glycosphingolipid synthesis in the early phase of this pathogenic process were studied by measurements of the incorporation of labeled galactose into glycosphingolipids of rat liver. [1-14C]Galactose was injected 2 h after galactosamine administration and the specific radioactivities of the glycosphingolipid precursors, UCPgalactose and UDPglucose, were determined. The specific radioactivity of UDPgalactose, when integrated over the whole period of radioactive synthesis, was four times higher in the galactosamine-treated animals than in the controls; the corresponding ratio of UDPglucose was 0.85. The pattern of the glycosphingolipids isolated from the livers of normal and galactosamine-treated rats resembled that described by Siddiqui and Hakomori (1970, Cancer Res. 30, 2930-2936); GL1, GM3,GM1, GD1, and a small amount of GT could be characterized. The specific radioactivities of glucose and galactose obtained from individual glycosphingolipids were determined in normal and galactosamine-treated livers. The synthesis of the glycosphingolipids was calculated using the respective data of the UDPhexoses. The labeling of glucosylceramide (GL1) was not altered and only a small change of GM3 could be detected; the synthesis of gangliosides GM1 and GD1, however, was inhibited by 95% or more between 4 and 6 h after galactosamine administration.