The regulation of carcinoembryonic antigen (CEA) expression by recombinant human interferon-γ (IFN-γ) was studied in a series of 7 human colorectal tumor cell lines at various stages of differentiation. Two of the colorectal cell lines were poorly differentiated and did not constitutively express CEA. IFN-γ treatment, however, induced CEA expression in one of those lines (i.e., DLD-1) as evidenced by the appearance of CEa-related mRNA transcripts, as well as the cell surface expression of the antigen as measured by flow cytometry and radioimmunoassay. In the highly differentiated colorectal tumor cell lines, IFN-γ treatment resulted in no detectable change in CEA content in whole cell extracts or in the percentage of cells positive for cell surface CEA expression. In fact, IFN-γ treatment of the highly differentiated LS174T cell line not only failed to alter CEA expression, but also failed to induce class II human leukocyte antigen expression. Therefore, the highly differentiated LS174T cell line and the poorly differentiated MIP cell line represent colorectal tumor cell types that are unresponsive to the ability of IFN-γ to induce alterations in tumor (i.e., CEA) or normal (i.e., class I and class II human leucocyte antigen) surface antigen expression. The most responsive of human colorectal tumor cells to the ability of IFN-γ to alter CEA expression were the moderately differentiated cell lines (i.e., HT-29, WiDr, etc.). IFN-γ treatment of those cell types increased the CEA content in cell extracts by 300–400%, and increased the percentage of cells positive for surface CEA expression from 30–45% to >80%. The effect of IFN-γ treatment on 2′,5′-oligoadenylate synthetase (2′-5′ A) activity was also studied using 4 of the 7 colorectal cell lines. Constitutive 2′-5′ A activity varied approximately 14-fold and was not correlated with degree of cellular differentiation. IFN-γ treatment increased 2′-5′ A activity in all 4 colorectal tumor cells tested. In particular, the ability to enhance 2′-5′ A activity in the MIP and LS174T cells, 2 colorectal tumor cell types that previously were shown to be unresponsive to IFN-γ-mediated changes in their antigenic phenotype, clearly separates cellular events regulating 2′-5′ A activity from those involved in regulating cell surface antigen expression. The findings also suggested that the regulation of CEA expression by IFN-γ is not related to the degree of cellular differentiation and, furthermore, provide some insight into which human tumor cell populations may be the most amenable to tumor antigen augmentation by IFN-γ in an adjuvant setting with a monoclonal antibody.