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Elements associated with the 35S promoter involved in generating the pregenomic RNA (35S RNA) of the pararetrovirus cauliflower mosaic virus have been extensively studied in heterologous systems, but little is known about their role in viral pathogenicity. To investigate these elements, premature termination codons were progressively inserted into the 3 end of the adjacent gene VI to dissect it from colinear 35S enhancer sequences. The ability to cause a systemic infection in plants was retained with loss of up to 40 amino acids from the gene VI polypeptide, but truncations into a putative zinc finger proved lethal. In the 35S promoter, removal of the TATA box also abolished infectivity. However, upstream deletions encompassing the 35S enhancer showed that the sequence between ؊207 and ؊56 from the cap site comprised nonessential elements, although complete removal of this fragment caused loss of infectivity even when domain spacing was restored by linker insertion. Two separate enhancer domains (؊207 to ؊150 and ؊95 to ؊56) were identified, of which either one or the other, but not both, was required for infectivity. Some mutations affected the cellular levels of viral RNAs in unexpected ways, as with removal of the as-1 enhancer element causing an increase in 35S RNA. Others altered the relative abundance of nuclear and cytoplasmic viral DNAs. Mutations in promoter domains thought to be involved in regulating tissue-specific expression did not significantly affect virus accumulation in leaves versus roots, whereas gene VI mutants showed reduced root accumulation. We conclude that elements associated with the cauliflower mosaic virus 35S promoter contain extensive nonessential regions that can behave differently in their proper context than as isolated elements.