Evidence has been obtained for the glycosylation of simian virus 40 (SV40) T antigen in SV40-infected TC7 cells. Both [3H]mannose and [3H]glucosamine are incorporated into T antigen in cells grown and labeled in medium containing fructose instead of glucose. In addition, T antigen is visualized by a carbohydrate stain specific for mannose and/or glucose residues. Finally, lectin binding studies suggest that T antigen contains galactose and/or galactosamine, since T antigen is specifically eluted from soybean lectin by 0.2 M galactose. When gel-purified, [3H]glucosamine-labeled T antigen is subjected to tryptic peptide mapping, label is found in only one peptide, thought to correspond to the methionine-containing peptide extending from Asn-653 to Arg-691, near the carboxy-terminal end of T antigen. Insensitivity to tunicamycin and the localization of the glycosylation site in the carboxy-terminus of T antigen, and not at Asn-153, suggest that T antigen is not N-glycosylated. Cell fractionation studies show that [3H]glucosamine-labeled T antigen is preferentially associated with the nuclear matrix of SV40-infected TC7 cells.