The insecticidal Bacillus thuringiensis Cry1Ac delta-endotoxin specifically binds to a 120 kDa aminopeptidase N (APN) in the midgut of susceptible insects such as Manduca sexta, Heliothis virescens, Lymantria dispar and Plutella xylostella. The 120 kDa APN has a glycosylphosphatidylinositol (GPI) anchor susceptible to the action of GPI-specific phospholipase C (PIPLC). Here we show that Cry1Ac pore-forming activity depends on the amount of APN present on brush border membrane vesicles (BBMV) from Trichoplusia ni larvae. Inhibition of APN activity with bestatin did not affect Cry1Ac pore formation, suggesting that Cry1Ac action depends on the presence of APN, but not on its enzymatic activity. N-Acetyl-D-galactosamine blocks the action of the toxin, indicating that this sugar is also directly involved in the Cry1Ac toxin-receptor interaction. Membrane potential measurements using PIPLC treated and non-treated BBMV suggest that both APN could participate as Cry1Ac receptor. The kinetic characterization of PIPLC sensitive and resistant APN indicates that they could be different isoforms. Finally, we show that in the presence of 200 mM Cs+ intrinsic BBMV T. ni channel permeability is not observed, while the toxin induced permeability is not affected, allowing an accurate analysis of the effect of the Cry1Ac toxin on T. ni midgut membranes.