In order to investigate the function of the 29K protein of tobacco rattle virus (TRV), we introduced different mutations in the 29K protein gene and analyzed the biological properties of the subsequent transcripts in tobacco plants. Although none of the mutant RNAs was able to accumulate to a detectable level, the defects in the 29K protein could be complemented by coinoculation with wild-type TRV or tobacco mosaic virus (TMV). Complementation was also achieved in transgenic plants expressing the homologous TMV 30K protein which is involved in cell-to-cell movement, but without inducing distinctive symptoms. Transcripts of chimeric TRV clones containing duplicate genes for the 29K protein initiated infections with formation of necrotic lesions and the progeny retained only one copy of the gene. These experiments demonstrate that the 29K protein is not required for viral RNA replication and, because the TRV transcripts do not encode the coat protein, that the 29K and 30K proteins act on nonencapsidated RNA. In addition to potentiating viral movement, the TRV 29K protein may also play a role in symptom induction on tobacco.