Imidazolide derivatives of long-chain fatty acids are shown to be potent irreversible activators of bee venom phospholipase A2. Activation corresponds to the addition of a single acyl residue to the protein molecule. The rate of activation increases with the acyl chain length of the activator, but the highest activation factors are given by the oleoyl and linoleoyl residues. Acyl group activation of the enzyme substitutes completely for activation by free fatty acids (but produces 3--4-fold higher activities) indicating that fatty acids are allosteric activators of the enzyme. The degree of activation is calcium dependent and exceeds 100-fold at low calcium concentration. Activation is extremely sensitive to substrate structure, but modification of the substrate surface by intercalated activator does not form the basis of this specificity.