Using specific antisera raised against synthetic peptides, we find that three distinct GTP-binding protein alpha subunits remain bound to the plasma membrane even after activation with nonhydrolyzable GTP analog. Trypsin cleaves each alpha subunit at a site near the amino-terminus and quantitatively releases the large fragment from the membrane. Previous results indicated that alpha subunits are essentially cytoplasmic proteins tethered to the inner surface of the membrane via an amino-terminal stalk.