Activities of the enzymes of the monosialoganglioside pathway [lactosylceramide → N-acetylneuraminic acid (sialic acid) (NAN)-galactose (Gal)-N-acetylgalactosamlne (GalNAc)-(NAN)-Gal-glucose (Glc)-ceramide (Cer) (GD1a)] were elevated progressively above those of control liver during tumorigenesis induced by N-2-fluorenylacetamide in precancerous tissues and well-differentiated hepatomas. The activities of UDP-Gal:GalNAc-(NAN)-Gal-Glc-Cer (GM2) galactosyltransferase and, to a lesser extent, CMP-NAN:Gal-GalNAc-(NAN)-Gal-Glc-Cer (GM1) sialyltransferase were correlated directly with early tumor growth. Activities of the first enzyme in the biosynthetic sequence, the CMP-NAN:lactosylceramide sialyltransferase, were elevated 2.5-fold in precancerous liver tissues and hyperplastic nodules and remained near that level in well-differentiated and poorly differentiated hepatomas. The UDP-GalNAc:NAN-Gal-Glc-Cer (GM3) N-acetylgalactosaminyltransferase was elevated in tissues surrounding nodules and hepatomas as well as in well-circumscribed, poorly differentiated hepatomas, but it was depressed in nodules. CMP-NAN was not incorporated into GD1a in any tissue. In the various types of tumors, the relative levels of activities of blosynthetic enzymes of the monosialoganglioside pathway correlated with the glycolipid levels in this branch of the pathway. Normal rat liver contained high levels of the enzymes of the disialoganglioside pathway in which tetrasialoganglioside is synthesized from hematoside via disialoganglioside intermediates. Specific activities of the branch-point enzyme CMPNAN: GM3 sialyltransferase were generally significantly lower than those of control livers in all hepatoma types, and a loss of CMP-NAN: Gal-GalNAc-(NAN)2-Gal-Glc-Cer (GD1b) sialyltransferase was observed in two of three well-circumscribed, poorly differentiated hepatomas. The specific activities of other biosynthetic enzymes of the disialoganglioside pathway, UDP-GalNAc:(NAN)2-Gal-Glc-Cer N-acetylgalactosaminyltransferase and UDP-Gal:GalNAc(NAN)2-Gal-Glc-Cer galactosyltransferase, were either near or above control activities in these same tumors. Deficiency of the branch-point enzyme may be a potentially important contributor to the progressive rise in the ratio of GM1 + GD1a to GD1b + GT [NAN-Gal-GalNAc-(NAN)2-Gal-Glc-Cer or (NAN)3-Gal-Glc-Cer] characteristic of hepatomas. These findings suggested that glycolipid alterations during tumorigenesis are a direct reflection of alterations in the activities of their respective biosynthetic enzymes.