Rhizobium leguminosarum colonizes host cells and tissues through infection threads, which are tubular in-growths of the plant cell wall. Monoclonal antibody MAC265 recognizes a plant matrix glycoprotein (MGP) associated with the lumen of these infection threads. This glycoprotein is also released in soluble form from the root tips of pea seedlings. In the presence of hydrogen peroxide, release of glycoprotein from root tips was not observed. Extractability from root tips was therefore used as the basis for investigating the peroxide-driven insolubilization of MGP and the possible involvement of two extracellular enzymes, peroxidase (POD) and diamine oxidase (DAO), was investigated. Release of MGP from root tips was enhanced by application of POD and DAO inhibitors (salicylhydroxamic acid and o-phenanthroline, respectively). Furthermore, release of MGP was inhibited by pretreatment of roots with putrescine (the substrate of DAO) and also by application of a partially purified extract of DAO from pea shoots. Following inoculation of pea roots with R. leguminosarum, elevated levels of DAO transcript were observed by reverse transcriptase-polymerase chain reaction (RT-PCR), but these then dropped to a low level from 4 to 10 days post inoculation, rising again in more mature nodules. In situ hybridization studies indicated that the bulk of the transcription was associated with the infected tissue in the center of the nodule. On the basis of these observations, we postulate that DAO may be involved in the peroxide-driven hardening of MGP in the lumen of infection threads and in the intercellular matrix.