We isolated and sequenced the two major tRNAGln isoacceptors with CUG and UmUG anticodons from the cytoplasm of Nicotiana rustica. These are the first tRNAsGln of nuclear origin characterized in plants. The tRNAGln sequences were used to design probes for the isolation of the corresponding genes from a nuclear DNA library of N. rustica. The two cloned Nicotiana tRNAGln genes, coding for either of the two isoacceptors, are efficiently transcribed in HeLa cell nuclear extract. In vitro translation in the presence of purified Nicotiana tRNAsGln was carried out in a wheat germ extract partially depleted of endogenous tRNAs. Cytoplasmic (cyt) tRNAGlnCUG and to a lesser extent cyt tRNAGlnUmUG stimulated readthrough over the UAG stop codon present in the tobacco mosaic virus-specific context. The two tRNAGln isoacceptors are the second class of natural UAG suppressors identified in plants, in addition to cyt tRNATyrGΨA which has previously been characterized as the first natural UAG suppressor.