Mucins are among the best described human tumor-associated antigens. At least 73 tumor-reactive anti-mucin antibodies have been described; in addition, we have previously demonstrated the existence of tumor-specific cytotoxic T-lymphocyte epitopes on the mucin produced by breast and pancreatic tumors. To determine whether the appearance of tumor-associated mucin epitopes can be explained by altered post-translational modification of mucin in tumors, or whether the generation of these epitopes requires changes in the mucin gene itself, we studied four Burkitt's lymphomas and six Epstein-Barr virus-immortalized B-cell lines transfected with an expression construct containing the mucin complementary DNA. Transfected cell lines showed stable maintenance of the mucin gene, which comprises 20 or more tandem repeats of a 60-nucleotide sequence. Transfected cells expressed many tumor-associated mucin epitopes, suggesting that the changes in mucin synthesis seen in breast and pancreatic tumors are present in other malignant cell types as well. Furthermore, even though each cell line was transfected with the identical mucin construct, each expressed a different subset of tumor-associated mucin epitopes. This suggests that the specificity of these epitopes for tumors is not due to genetic alterations of the mucin gene in tumors. Incubating transfected cells with phenyl-N-acetyl-alpha-galactosaminide, an inhibitor of O-linked glycosylation, altered cell surface carbohydrate structures and resulted in increased expression of all tumor-associated epitopes, implicating incomplete glycosylation of mucin in the generation of these epitopes. These findings suggest that alterations in the posttranslational modification of normal gene products can result in the expression of novel epitopes. Furthermore, the ability to transfect cancer patients' Epstein-Barr virus-immortalized B-cell lines with mucin will provide an unlimited supply of autologous, mucin-bearing cells with which to study these patients' T-cell response to mucin.