We have isolated and sequenced chloroplast (chl) and cytoplasmic (cyt) cysteine tRNAs from Nicotiana rustica. Both tRNAs carry a GCA anticodon but beyond that differ considerably in their nucleotide sequences. One obvious distinction resides in the presence of N6-isopentenyladenosine (i6A) and 1-methylguanosine (m1G) at position 37 in chl and cyt tRNA(Cys) respectively. In order to study the potential suppressor activity of tRNAs(Cys) we used in vitro synthesized zein mRNA transcripts in which an internal UGA stop codon had been placed in either the tobacco rattle virus (TRV)- or tobacco mosaic virus (TMV)-specific codon context. In vitro translation was carried out in a messenger- and tRNA-dependent wheat germ extract. Both tRNA(Cys) isoacceptors stimulate read-through over the UGA stop codon, however, chl tRNA(GCA)Cys is more efficient than the cytoplasmic counterpart. The UGA in the two viral codon contexts is suppressed to about the same extent by either of the two tRNAs(Cys), whereas UGA in the beta-globin context is not recognized at all. The interaction of tRNA(GCA)Cys with UGA requires an unconventional G:A base pair in the wobble position, as postulated earlier for plant tRNA(G psi A)Tyr misreading the UAA stop codon. This is the first case that a cysteine-accepting tRNA has been characterized as a natural UGA suppressor.