We have investigated the molecular basis for the reported synergism between peptaibols and cell wall hydrolytic enzymes in the antagonism of phytopathogenic fungi by Trichoderma harzianum. beta-Glucan synthase activity on isolated plasma membranes of Botrytis cinerea was inhibited in vitro by the peptaibols trichorzianin TA and TB, and this inhibition was reversed by the addition of phosphatidylcholine. beta-Glucan synthesis in vivo, assayed by the incorporation of [2-(3)H]glucose into cell wall material, was inhibited by the presence of peptaibols, and this inhibition was synergistic with exogenously added T. harzianum beta-1,3-glucanase. This synergism is therefore explained by an inhibition of the membrane-bound beta-1,3-glucan synthase of the host by the peptaibols, which inhibit the resynthesis of cell wall beta-glucans, sustain the disruptive action of beta-glucanases, and all together enhance the fungicidal activity. Therefore, we have identified cell wall turnover as a major target of mycoparasitic antagonism.