Citrus tristeza virus (CTV), a member of the closterovirus group, is one of the more complex single-stranded RNA viruses. The 5' portion of its 19,296-nt, single-stranded RNA genome is expressed as an approximately 400-kDa polyprotein that is proteolytically processed, while the 10 3' open reading frames are expressed from 3'-coterminal subgenomic RNAs (sg RNAs). As an initial examination of the gene expression of this virus, we found that the kinetics of accumulation of genomic and sg RNAs and coat protein of the T36 isolate of CTV were similar in protoplasts of the natural host, citrus, and the experimental nonhost Nicotiana benthamiana. Newly synthesized genomic RNA was detected 2 days postinoculation and increased to a maximum at 3-5 days. The RNA complementary to the full-length virion RNA increased with similar kinetics, but at approximately one-tenth the concentration of genomic plus strands. Most of the abundant sg RNAs also accumulated in parallel to that of the genomic RNA. However, the smallest sg RNA, which corresponds to the p23 gene, increased earlier. The different sg RNAs accumulated in greatly differing amounts, in general with 3'-most sg RNAs accumulating to higher levels than 5' sg RNAs. However, some 3' sg RNAs (p13 and p18) accumulated to low levels. The two 3'-most sg RNAs (p23 and p20) accumulated to high levels approximately equal to that of the genomic RNA. The accumulation curve for coat protein paralleled that of its mRNA, suggesting that its regulation was transcriptional. Progeny virions from protoplasts were used to sequentially infect new protoplasts, serving as a potential source of virus that could evolve free from the genetic selection in intact plants for aphid transmission and movement.