The cauliflower mosaic virus (CaMV) 35 S RNA is a full-length transcript of the viral genome. It encodes the genes VII and I-V, arranged in tandem along the RNA, preceded by a long leader region (600 bases) containing many short open reading frames. We have examined the effects of the leader and the first gene (gene VII) on downstream gene I translation in vitro and in an in vivo transient expression system (carrot protoplasts). RNAs from constructs containing the intact leader, and from various deletion constructs, were translated in a rabbit reticulocyte system. Gene I was translated efficiently only when the long leader region and the upstream gene VII were deleted. Translational fusions of gene VII or I to the firefly luciferase reporter gene were also constructed, and a similar series of leader sequence deletion mutants were examined in vivo and in vitro. The 600-base leader region was found to repress translation of gene VII 8- to 30-fold as compared to the truncated gene lacking the leader region. Gene I expression as compared to that of gene VII was reduced an additional 7- to 20-fold by the presence of the upstream leader region including gene VII. This represented an overall reduction in gene I expression of greater than 100-fold as compared to expression in the absence of any leader sequence. The reduced translation of gene I in the context of the 35 S RNA leader region was not due to the action of the gene VII protein product but may result from efficient blocking of scanning 40 S ribosomes by translation of upstream open reading frames.