The NCA cDNA, which represents a gene belonging to the CEA family, was inserted into an SV40 early promoter-driven expression vector and used for transfection of mouse NIH/3T3 cells. A cell line, NIH/3T3/KNCA IG7, was selected which expressed a molecule with an apparent molecular weight of 110,000. The mode of membrane attachment of this NCA, which we already proposed to be anchored via glycosyl-phosphatidylinositol, was investigated by treatment of NIH/3T3/KNCA IG7 cells with phosphatidylinositol-specific phospholipase C from Bacillus thuringiensis. Two independent methods, flow cytometry and immunoprecipitation of [3H]-labelled surface glycoproteins, clearly demonstrated that the NCA molecule expressed by NIH/3T3/KNCA IG7 cells is indeed anchored into the membrane via glycosyl-phosphatidylinositol. Furthermore, these results support our previous biochemical data on NCA-50, by unequivocally showing that the NCA cDNA used for transfection encodes an NCA molecule related to NCA-50 and NCA-90.