The in vitro phosphorylation by [gamma-32P]ATP of a 34-kDa plasma membrane-associated protein (pp34) from tomato and potato is strongly enhanced in the presence of alpha-1,4-D-polygalacturonic acid (PGA) fragments (Farmer, E. E., Pearce, G., and Ryan, C. A. (1989) Proc. Natl. Acad. Sci. U. S. A. 86, 1539-1542) that activate the expression of defensive and developmental genes in plant tissues. [gamma-35S]ATP, but not [gamma-35S]GTP, has now been found to strongly label pp34 in the presence of the PGA fragments. PGA-enhanced phosphorylation of pp34 is at one or more threonine residue(s) and therefore is the product of a serine/threonine kinase. alpha-1,4-L-Polyguluronic acid (PGU) enhances thiophosphorylation of pp34, but is less effective than PGA. beta-1,4-D-Polymannuronic acid (PMA) is inactive. In vivo synthesis of proteinase inhibitors in tomato leaves in response to PGA, PGU, and PMA parallels enhancing activities in in vitro phosphorylation assays. The minimum oligogalacturonide lengths that enhance in vitro thiophosphorylation of pp34 are about 14-15 residues, which are near the minimum sizes of uronides required to elicit a variety of localized defensive and developmental responses in plants. The lengths of biologically active galacturonic acid oligomers are of the same length that form strong intermolecular complexes in solution with Ca2+. Uronide-Ca2+ complexes are proposed to be the active molecular species that initiate the signal transduction pathways regulating uronide-responsive genes.