The Pto gene encodes a serine/threonine kinase that confers resistance to bacterial speck disease in tomato. Using the yeast two-hybrid system, we identified a second serine/threonine kinase, Pto-interacting 1 (Pti1), that physically interacts with Pto. Cross-phosphorylation assays revealed that Pto specifically phosphorylates Pti1 and that Pti1 does not phosphorylate Pto. Fen, another serine/threonine kinase from tomato that is closely related to Pto, was unable to phosphorylate Pti1 and was not phosphorylated by Pti1. Expression of a Pti1 transgene in tobacco plants enhanced the hypersensitive response to a P. syringae pv. tabaci strain carrying the avirulence gene avrPto. These findings indicate that Pti1 is involved in a Pto-mediated signaling pathway, probably by acting as a component downstream of Pto in a phosphorylation cascade.