Botulinum neurotoxin type A (BoNT/A) is largely employed in human therapy because of its specific inhibition of peripheral cholinergic nerve terminals. BoNT/A binds to them rapidly and with high specificity via its receptor binding domain termed HC. Recent evidence indicate that BoNT/A interacts specifically with polysialogangliosides and with a luminal loop of the synaptic vesicle protein SV2 via the C-terminal half of HC. Here we show that the N-terminal half of HC binds to sphingomyelin-enriched membrane microdomains and that it has a defined interaction With phosphatidylinositol phosphates (PIP). We have identified a PIP binding site in this half of HC and we show how this interaction Could predispose BoNT/A for membrane insertion, which is the step subsequent to binding, in the four-steps route leading BoNT/A inside nerve terminals. (C) 2009 Elsevier Inc. All rights reserved.