Botulinum Neurotoxin (BoNT) is one of the most potent toxins known to human kind. The Atomic Force Microscope (AFM) was employed to investigate the conditions under which BoNT type A heavy chain would bind and/or insert into mica supported dipalmitoylphosphatidylcholine (DPPC) lipid bilayers. As an alternate technique, DPPC/GT1b or total ganglioside extract (80:20) monolayers of a Langmuir Blodgett (LB) Trough were adapted to be artificial membrane models for toxin insertion studies. We conclude that LB monolayer studies are a promising candidate for BoNT/A membrane insertion investigation. Botulinum neurotoxin serotype A insertions into the LB monolayers in the presence of BoNT/A low affinity ganglioside receptor alone, independent of pH. This thermodynamic evidence indicates that BoNT/A may begin its heavy chain insertion into the cholinergic nerve ending before endocytosis and acidification.