The umbrella and penknife models hypothesize that insecticidal Bacillus thuringiensis Cry toxins partition into the apical membrane of the insect midgut by insertion of only two alpha-helices from domain I of the protein, alpha-helices 4 and 5 in the case of the umbrella model and alpha-helices 5 and 6 in the case of the penknife model. Neither model envisages membrane partitioning by domains II and III. In this study, we present data suggesting that mutations in the domain II residue, F371, affect insertion of the whole toxin into Manduca sexta brush border membrane vesicles (BBMVs). Using steady state fluorescence measurements combined with a proteinase K protection assay, we show that mutants of F371 have lost their ability to insert into the BBMV, even though binding to cadherin is almost unaffected. The study also identifies a difference in partitioning of toxins into artificial lipid vesicles (SUVs) as opposed to native BBMVs. While the F371 mutations block insertion of domains I and II into BBMVs, they only block domain II insertion into SUVs. Bioassay and voltage clamping of midguts also confirm the fluorescence data that the noninserting mutants are nontoxic. Our study leads us to propose that, in contrast to previous models of individual free helices inserting into the membrane, the toxin enters into the membrane as a whole molecule or oligomers of the molecule, wherein the domain II residue F371 has a vital role to play in membrane insertion.