Antimicrobial peptides (So-D1-7) were isolated from a crude cell wall preparation from spinach leaves (Spinacia oleracea cv. Matador) and, judged from their amino acid sequences, six of them (So-D2-7) represented a novel structural subfamily of plant defensins (group IV). Group-IV defensins were also functionally distinct from those of groups I-III. They were active at concentrations < 20 microM against Gram-positive (Clavibacter michiganensis) and Gram-negative (Ralstonia solanacearum) bacterial pathogens, as well as against fungi, such as Fusarium culmorum, F. solani, Bipolaris maydis, and Colletotrichum lagenarium. Fungal inhibition occurred without hyphal branching. Group-IV defensins were preferentially distributed in the epidermal cell layer of leaves and in the subepidermal region of stems.