The cadherin-like protein Bt-R₁ is a Cry1A-binding protein on larval midgut epithelia of Manduca sexta. The extracellular portion of Bt-R₁ is composed of 12 cadherin repeats (CR) and a membrane-proximal domain (MPED). Expression of truncated Bt-R₁ proteins on the surface of Drosophila S2 cells demonstrated that the minimum fragment necessary for toxin binding and cytotoxicity was the CR12-MPED region. The cDNA fragment coding CR12-MPED was expressed in Escherichia coli and purified. In vitro, this CR12-MPED peptide competed with Cry1Ab binding to Bt-R₁ expressed in Drosophila S2 cells or to midgut brush border membrane vesicles from M. sexta larvae. Unexpectedly, insect bioassays indicated that the mixture of Cry1Ab and CR12-MPED peptide (mass ratios 1:1, 1:10, and 1:100) did not block Cry1Ab toxicity to M. sexta larvae, but significantly enhanced toxin-induced mortality. No enhanced toxicity was detected in control bioassays with CR12-MPED peptide alone. Enhanced toxicity by CR12-MPED peptide was also observed using Cry1Ac against M. sexta, Heliothis virescens, and Helicoverpa zea larvae. Microscopy showed that the mixture of Cry1Ab or Cry1Ac with CR12-MPED peptide did not change the localization of toxin binding on M. sexta larval midgut. However, when mixed with CR12-MPED peptide, Cry1Ab formed large aggregates at its binding sites, whereas such toxin aggregates were not observed for Cry1Ac. The potential mechanism resulting in enhanced Cry1A toxicity by CR12-MPED peptide will be discussed.