Membrane cofactor protein (MCP) cDNA was transfected into Chinese hamster ovarian tumor (CHO) cells and the functional properties of the expressed protein were studied. Cells adherent to flasks were essential for continuous expression of MCP on CHO cells. If the cells were maintained in noncoated flasks, MCP expression was markedly reduced but upon being transferred to coated flasks reexpressed the protein. MCP expressed on CHO cells had the expected m.w., approximately 50 kDa, and possessed factor I-cofactor activity. By a propidium iodide incorporation assay and by 51Cr release assay, antibody-sensitized CHO cells expressing MCP were protected from C-mediated cytotoxicity. The inhibition of lytic activity correlated with a decrease in C3 deposition. This host cell protective activity was exerted efficiently for the alternative pathway. The classical pathway was not blocked by MCP unless the cells were presensitized with low concentrations of antibody. These results imply that MCP primarily protects host cells from alternative pathway-mediated C3 targeting. In a pathologic state such as autoimmune diseases, the binding of an autoantibody to a target may overcome this protective effect of MCP via the classical pathway.