To develop an antiviral agent and virus-resistant plants, a cDNA clone encoding Phytolacca insularis antiviral protein (PIP) was isolated from a cDNA library constructed with poly(A)+ RNA purified from leaves of P. insularis. The PIP cDNA contains an open reading frame encoding 307 amino acids. The deduced amino acid sequence includes a putative signal sequence of 22 amino acids at the N-terminus. The amino acid sequence of PIP shares 84% homology with that of the pokeweed antiviral protein (PAP). In addition, the mature PIP exhibits the conserved putative active site found in other ribosome-inactivating proteins (RIPs). Recombinant PIP (rPIP) synthesized in Escherichia coli inhibits protein synthesis in vitro in rabbit reticulocyte lysate through the N-glycosidase activity in a similar manner with other RIPs. Local lesion assays with purified rPIP revealed that it inhibits infection of various viruses to plants. Transgenic potato plants expressing the PIP cDNA under the control of the cauliflower mosaic virus 35S promoter are resistant to viruses, such as potato virus X, potato virus Y, and potato leafroll virus. These results suggest that the PIP cDNA could be used for the development of an antiviral agent and transgenic plants resistant against a broad spectrum of plant viruses infecting through both mechanical and aphid transmission.