Annexins are Ca2+-dependent phospholipid-binding proteins composed of two domains: A conserved core that is responsible for Ca2+- and phospholipid-binding, and a variable N-terminal tail. A Ca2+-independent annexin S-membrane association has been shown to be modulated by the presence of cholesterol in the membranes. Herein, the roles of the core and the N-terminal tail on the cholesterol-enhancement of annexin 2 membrane binding and aggregation were studied. The results show that (i) the cholesterol-mediated increase in membrane binding and in the Ca2+ sensitivity for membrane aggregation were not modified by a N-terminal peptide (residues 15-26), and were conserved in mutants of the N-terminal end (S11 and S25 substitutions); (ii) cholesterol induced an increase in the Ca2+-dependent membrane binding and aggregation of the N-terminally truncated protein (Delta 1-29); and (iii) annexins 5 and 6, two proteins with unrelated N-terminal tails and homologous core domains showed a cholesterol-mediated enhancement of the Ca2+-dependent binding to membranes. These data indicate that the core domain is responsible for the cholesterol-mediated effects. A model for the cholesterol effect in membrane organisation, annexin binding and aggregation is discussed.