A cDNA clone, NA-PI-II, encoding a protein with partial identity to proteinase inhibitor (PI) II of potato and tomato has been isolated from a cDNA library constructed from Nicotiana alata stigma and style mRNA. The cDNA encodes a polypeptide of 397 amino acids with a putative signal peptide of 29 amino acids and six repeated domains, each with a potential reactive site. Domains 1 and 2 have chymotrypsin-specific sites and domains 3, 4, 5, and 6 have sites specific for trypsin. In situ hybridization experiments demonstrated that expression of the gene is restricted to the stigma of both immature and mature pistils. Peptides with inhibitory activity toward chymotrypsin and trypsin have been isolated from stigmas of N. alata. The N-terminal amino acid sequence obtained from this protein preparation corresponds to six regions in the cDNA clone NA-PI-II. The purified PI protein preparation is likely to be composed of a mixture of up to five similar peptides of approximately 6 kD, produced in vivo by proteolytic processing of a 42-kD precursor. The PI may function to protect the reproductive tissue against potential pathogens.