To determine the possibility that calmodulin, a cytosolic regulatory protein, and ammodytoxin, a neurotoxic secreted phospholipase A2, interact in vivo, we studied the dependence of their association on Ca 2+. The interaction between the two proteins was positively dependent on Ca2+, and greatest at millimolar concentrations of this ion. Importantly, they interacted already in the presence of sub-micromolar concentrations of Ca2+, as demonstrated by affinity labelling, laser tweezers and surface plasmon resonance. Tight binding of the secreted phospholipase A2 to calmodulin is therefore expected on depolarization of the axolemma, when the concentration of free Ca2+ at active zones rises to 100 μM. These results strengthen the proposal that binding of ammodytoxin to calmodulin is a step in the process of presynaptic toxicity of this and related phospholipases A2. Moreover, they suggest that some other (patho)physiological effects induced by endogenous secreted phospholipases A2 could be also due to their interaction with calmodulin in the cytosol of cells.