Three cysteine proteinase inhibitor cDNA clones (pL1, pR1, and pN2) have been isolated from a soybean (Glycine max L. Merr.) embryo library. The proteins encoded by the clones are between 60 and 70% identical and contain the consensus QxVxG motif and W residue in the appropriate spatial context for interaction with the cysteine proteinase papain. L1, R1, and N2 mRNAs were differentially expressed in different organs of plants (juvenile and mature) and seedlings, although N2 mRNA was constitutive only in flowers. R1 and N2 transcripts were induced by wounding or methyl jasmonate (M-JA) treatment in local and systemic leaves coincident with increased papain inhibitory activity, indicating a role for R1 and N2 in plant defense. The L1 transcript was constitutively expressed in leaves and was induced slightly by M-JA treatment in roots. Unlike the chymotrypsin/trypsin proteinase inhibitor II gene (H. Peña-Cortés, J. Fisahn, L. Willmitzer [1995] Proc Natl Acad Sci USA 92: 4106-4113), expression of the soybean genes was only marginally induced by abscisic acid and only in certain tissues. Norbornadiene, a competitive inhibitor of ethylene binding, abolished the wounding or M-JA induction of R1 and N2 mRNAs but not the accumulation of the wound-inducible vspA transcript. Presumably, ethylene binding to its receptor is involved in the wound inducibility of R1 and N2 but not vspA mRNAs. Bacterial recombinant L1 and R1 proteins, expressed as glutathione S-transferase fusion proteins, exhibited substantial inhibitory activities against vicilin peptidohydrolase, the major thiol endopeptidase in mung bean seedlings. Recombinant R1 protein had much greater cysteine proteinase inhibitor activity than recombinant L1 protein, consistent with the wound inducibility of the R1 gene and its presumed role in plant defense.