The ganglioside pattern of both SV40- and polyoma virus-transformed mouse cell lines differs from that of the parent cell lines or of cell lines that have transformed spontaneously in tissue culture. This is manifested by a dramatic decrease of gangliosides with an oligosaccharide chain larger than sialyllactose. Present investigations indicate that this change probably cannot be attributed to excessive catabolism of gangliosides, but is caused by impaired synthesis of tri- and tetrahexosyl gangliosides in the virus-transformed cell lines. We present evidence for the block of a required step for the biosynthesis of these ganglioside homologs. The block involves the enzyme catalyzing the transfer of N-acetylgalactosamine from uridine diphosphate N-acetylgalactosamine to hematosides (N-glycolylneuraminyl or N-acetylneuraminylgalactosylglucosyl ceramide). This well-defined enzymatic change opens the way for studies of the biochemical mechanism of the alteration of cell membranes which occurs after transformation by the tumorigenic DNA viruses polyoma and SV40.