Unlike other pathogens, plant viruses are hardly controlled by chemical agents. Potato virus Y (PVY) is distributed around the world, and causes a great loss economically. In an attempt to minimize the damage by viruses, the PVY coat protein (CP) gene was introduced into tobacco by Agrobacterium-mediated transformation. A significant proportion of the transgenic plants displayed resistance to PVY and showed substantially decreased CP transgene expression at both protein and steady-state mRNA levels compared to susceptible transgenic or nontransgenic plants. A resistant plant was selected and self-fertilized for several generations until T4 progenitor lines were obtained. Most of these T4 plants accumulated extremely low levels of CP protein and steady-state mRNA, and exhibited almost complete resistance to PVY. DNA gel blot analysis revealed that the transgenic plants typically had two or three copies of the transgene. These results are characteristic of pathogen-derived resistance, in which the resistance against virus is the consequence of post-transcriptional gene silencing directed by homologous transgenes. To uncover factors that may play roles in gene silencing, sequences in the 3' part of the transcribed region of the CP gene were transcribed in vitro and the RNA fragments were incubated with cell extracts from transgenic plants. A ribonuclease activity was detected that appeared to be specific for this transcript in the PVY-resistant transgenic plants.