Qa-2, a cell-surface glycoprotein anchored by phosphatidylinositol (PI), is structurally related to the class I transplantation antigens H-2 K, D, and L, which are integral membrane glycoproteins. The predicted transmembrane segment of Qa-2 differs from those of H-2 K, D, and L by the presence of an aspartate in place of a valine at position 295. A single base change that replaced this aspartate with valine resulted in cell-surface Qa-2 molecules that were insensitive to hydrolysis by a PI-specific phospholipase C and more resistant to papain cleavage, properties shared by H-2D. Cells expressing Asp → Val mutant Qa-2 proteins were still able to attach a PI anchor to endogenous proteins such as Thy-1 and J11D. It therefore appears that this single amino acid change converts Qa-2 from a PI-linked form into an integral membrane protein.