Thy-1 is a glycoprotein present on the membrane of murine cells of the T-lineage. The mature Thy-1 is anchored to the membrane via a glycolipid, phosphatidylinositide. In order to study the regulation of the synthesis and membrane insertion of this protein, the biochemical properties of a Thy-1.2 negative variant T-lymphoma cell (RL male 1.4) were studied. It contains intracellular Thy-1 protein but fails to express it on the cell surface. While the wild type and the mutant show similar labelling of the intracellular Thy-1 glycoprotein with amino acids, no ethanolamine is incorporated into the Thy-1 molecule of RL male 1.4. A plasmid, pT1, containing the normal Thy-1.2 gene and bacterial gpt gene was transfected into RL male 1.4 and into the murine plasmacytoma cell, J558L. A transfected plasmacytoma, T1J2, synthesized a normal sized Thy-1 protein and displayed the antigen on the membrane. In contrast, the mycophenolic acid resistant RL male 1.4 transfectants did not display Thy-1.2 on the cell surface, despite the presence of substantial amounts of Thy-1 intracellularly. Two other antigens known to be anchored in the membrane by phospholipid, Ly-6e and Qa-2, were also examined in RL male 1.4. RL male 1.4 did not express Ly-6e after alpha interferon induction. In addition, the expression of Qa-2 antigen was greatly diminished in RL male 1.4 in comparison to RL male 1.3. Thus, the defect in RL male 1.4 is not restricted to Thy-1.2, but includes other similarly anchored glycoproteins as well. This implies that the addition of phospholipid to core proteins is similar, if not identical, for all these molecules and that the RL male 1.4 cell lacks the capacity to from the lipid glycoprotein linkage required for the expression of these proteins on the cell surface.