Transgenic burley (cultivars KY14, NC3433-33, and TN90) and flue-cured (cultivar K326) tobacco with resistance to four necrotic isolates of potato virus Y (PVY) were constructed. These tobacco cultivars were transformed with a chimeric gene designed to express the coat protein (CP) from the necrotic Chilean isolate of PVY CP expression among R(0) plants was undetectable by indirect enzyme-linked immunosorbent assay and Western blot (immunoblot) analysis. Transgenic R(0) plants that harbored from one to five neomycin phosphotransferase II (NPT II) transgene loci were identified. Although the NPT II and the PVY CP chimeric genes were linked within a common T-DNA, no correlation was found between the number of NPT II and CP transgenes. Inoculation of 41 independent transgenic KY14 R(0) plants with PVY-Chilean identified eight resistant plants, while inoculation of 30 transgenic K326 Ro plants identified six resistant plants. Inoculation of 17 transgenic NC3433-33 R(0) plants with PVY-Chilean identified six resistant plants, while inoculation of 50 transgenic TN90 R(0) plants with PVY isolate VAM B resulted in nine resistant plants. Progeny derived from PVY-inoculated but symptomless Ro plants were also resistant to the Chilean, Europe H, M(s)N(r), N-Canada, and VAM B isolates of PVY, demonstrating sexual transmission of the transgenes that condition resistance. We discuss the ability of the PVY-Chilean CP gene to protect transgenic tobacco K326, KY14, NC3433-33, and TN90 from infection by four necrotic isolates of PVY. The flue-cured tobacco cultivar K326, which carries a gene simultaneously conferring resistance against root-knot nematode and susceptibility to PVY strain M(S)N(R), is protected against this strain when transformed with the PVY-Chilean CP gene.