Bacillus thuringiensis CryIA(c) toxin binds to aminopeptidase N (EC 3.4.11.2), a 120-kDa glycoprotein located on the Manduca sexta midgut brush border membrane. We report that the CryIA(c) binding protein is anchored in the membrane by a glycosyl-phosphatidylinositol (GPI) anchor, and that toxin also binds to the hydrophilic form of the protein released by phosphatidylinositol-specific phospholipase C (PIPLC). The 120-kDa toxin binding protein, along with other brush border membrane proteins, was solubilized in Triton X-114 and then fractionated by phase separation into the detergent-rich phase. After cleavage by PIPLC and a second Triton X-114 phase separation, GPI-anchored proteins were converted to a hydrophilic form that partitioned into the detergent-poor phase. Cleaved GPI-anchored proteins were detected by probing protein blots with a polyclonal antiserum against the cross-reacting determinant of trypanosomal variant surface glycoprotein. Proteins of 115, 110, 106, 72 and 60 kDa had cross-reacting determinants, and [ 125I]CryIA(c) recognized the 115-kDa protein. Exhaustive PIPLC digestion released approx. 61% of the 120-kDa toxin binding protein, 46% of the aminopeptidase and 96% of the alkaline phosphatase. These results indicate that the majority of the 120-kDa toxin binding aminopeptidase is anchored in the brush border membrane through a glycolipid structure.