Monoclonal antibodies with specificity for mucin-like antigens have shown great promise for the diagnosis and therapy of human cancer. Heterogeneity in the expression of mucin-like antigens by tumor and normal cells has been noted in several previous studies. An understanding of the nature of this heterogeneity has important implications for the diagnostic and therapeutic usefulness of antibodies to mucin-like antigens. We have studied the mechanism of variability in expression of epitopes on a mucin-like antigen defined by monoclonal antibodies W1, W5, and W9 in the lung carcinoma cell line, Calu-1. Using the fluorescence activated cell sorter and clonal analysis, we have demonstrated that intercellular variability in mucin antigen expression by Calu-1 cells can be explained in part by heritable variation in the tumor cell population. Clonal cell lines were isolated which differ greatly in levels of epitopes for all three mucin directed antibodies. Levels of all three epitopes showed significant variation between different clonal lines but in general were coordinately regulated. Differences in epitope expression between two lines studied in detail could be attributed to a dramatic difference in expression of a high molecular weight mucin-like glycoprotein. In immunoblotting experiments the binding of all three antibodies to this glycoprotein was affected by sodium periodate and/or neuraminidase treatment, suggesting that the antibodies recognize carbohydrate epitopes. Thus, heterogeneity in expression of mucin-like glycoprotein antigens can result from heritable variations which affect expression of multiple carbohydrate epitopes.