Three abundant proteins of approximate molecular weights of 22, 23, and 24 kilodalton (kD) were purified from potato (Solanum tuberosum L.) tubers by DEAE cellulose and CM-52 cellulose ion exchange column chromatography, electroelution, and high-pressure liquid chromatography (HPLC). Antibodies specific to the gel purified 22-kD protein were prepared. Immunoblot analysis showed that the 22-, 23-, and 24-kD proteins were immunologically related and that these proteins were present in tubers and as higher molecular weight forms in leaves, but were not detectable in stems, roots, and stolons. The ratios of amino acid composition of the three purified proteins were equivalent, and all three proteins had identical amino-terminal sequences that match the deduced amino acid sequence of an abundant tuber protein cDNA, p34021;The 22-kD potato tuber proteins were potent inhibitors of serine proteinases. Wound induction of the genes coding for the 22-kD potato tuber proteins also was detected at the RNA level. Two of the three purified proteins from the 22-kD family of potato tuber protein were effective inhibitors of both trypsin and chymotrypsin. The third purified protein with molecular weight of approximately 24 kD inhibited only trypsin activity. Comparison of the amino acid sequence of the putative reactive sites of several members of the proteinase inhibitors with the deduced sequence of the 22-kD protein revealed that the 22-kD protein contained sequences which could potentially possess "double-headed" sites of inhibition, one against trypsin and the other against chymotrypsin;Like other potato proteinase inhibitors, the genes coding for the 22-kD proteins were developmentally regulated in tubers and environmentally regulated in leaves. In leaves, transcripts of the 22-kD protein family were detected 6 h after wounding and were highest after 12 h in locally wounded leaves. The strongest induction occurred systemically in nonwounded leaves in response to mechanical wounding. Cross-hybridization of a cDNA, p34021, which codes for the 22-kD tuber protein, with proteinase inhibitor I, and II cDNAs and a second family of 20-kD potato tuber cDNAs revealed no cross-homology. Members of this second group of 20-kD potato tuber proteins also exhibited wound-induction in leaves at the RNA level. This study identified an entirely new group of potato proteinase inhibitors whose regulated expression is similar to proteinase inhibitors I and II.