Several toxins that act on animal cells present different, but specific, interactions with cholesterol or sphingomyelin. In the present study we demonstrate that HlyA (α-haemolysin) of Escherichia coli interacts directly with cholesterol. We have recently reported that HlyA became associated with detergent-resistant membranes enriched in cholesterol and sphingomyelin; moreover, toxin oligomerization, and hence haemolytic activity, diminishes in cholesterol-depleted erythrocytes. Considering these results, we studied the insertion process, an essential step in the lytic mechanism, by the monolayer technique, finding that HlyA insertion is favoured in cholesterol- and sphingomyelin-containing membranes. On the basis of this result, we studied the direct interaction with either of the lipids by lipid dot blotting, lysis inhibition and SPR (surface plasmon resonance) assays. The results of the present study demonstrated that an interaction between cholesterol and HlyA exists that seems to favour a conformational state of the protein that allows its correct insertion into the membrane and its further oligomerization to form pores.