Stenotrophomonas maltophilia strain W81, isolated from the rhizosphere of field-grown sugar beet, produced the extracellular enzymes chitinase and protease and inhibited the growth of the phytopathogenic fungus Pythium ultimum in vitro. The role of these lytic enzymes in the interaction between W81 and P. ultimum was investigated using Tn5 insertion mutants of W81 incapable of producing extracellular protease (W81M1), extracellular chitinase (W81M2) or the two enzymes (W81A1). Lytic enzyme activity was restored in W81A1 following introduction of a 15 kb cosmid-borne fragment of W81 genomic DNA. Incubation of P. ultimum in the presence of commercial purified protease or cell-free supernatants from cultures of wild-type W81, the chitinase-negative mutant W81M2 or the complemented derivative W81A1(pCU800) resulted in hyphal lysis and loss of subsequent fungal growth ability once re-inoculated onto fresh plates. In contrast, commercial purified chitinase or cell-free supernatants from cultures of the protease-negative mutant W81M1 or the chitinase-and protease-negative mutant W81A1 had no effect on integrity of the essentially chitin-free Pythium mycelium, and did not prevent subsequent growth of the fungus. in soil microcosms containing soil naturally infested by Pythium spp., strains W81, W81M2 and W81A1(pCU800) reduced the ability of Pythium spp. to colonize the seeds of sugar beet and improved plant: emergence compared with the untreated control, whereas W81A1 and W81M1 failed to protect sugar beet from damping-off. Wild-type W81 and its mutant derivatives colonized the rhizosphere of sugar beet to similar extents. It was concluded that the ability of S. maltophilia W81 to protect sugar beet from Pythium-mediated damping-off was due to the production of an extracellular protease.