Cowpea chlorotic mottle virus (CCMV) has a tripartite, positive strand RNA genome. Genomic RNA3 (2.2 kb) encodes the 3a nonstructural protein and the coat protein, which are dispensable for viral RNA synthesis in protoplasts, but required for systemic infection of whole plants. In protoplasts, portions of the 5' and intercistronic noncoding regions of CCMV RNA3 are also dispensable for RNA3 replication and for transcription of the subgenomic coat protein mRNA. To determine whether these noncoding sequences are required for systemic infection, a series of 5' and intercistronic deletions in RNA3 were tested for their effects on the infection of cowpea plants, a natural host of CCMV. The results refine the mapping of the subgenomic mRNA promoter and show that at least 144 bases in the 5' noncoding region and at least 125 bases in the intercistronic noncoding region of CCMV RNA3 are dispensable for systemic infection. For mutants with deletions within these regions, no differences were noted in the rate of infection spread, and the level of virus accumulation in systemically infected tissue 10-14 days postinoculation was 60-100% of wild type (wt). However, the largest viable intercistronic deletion transformed the nearly symptomless appearance of wt CCMV infections to an extensive, bright yellow chlorosis, showing that infection pathology can be altered by mutations with a regulatory rather than a protein-coding character. In addition, neither 5' nor intercistronic deletion mutants competed effectively with wt CCMV in whole plant co-infection experiments; i.e., such mutants were not detectable in systemically infected tissue after co-inoculation with wt CCMV. Thus, although substantial portions of both the 5' and the intercistronic noncoding regions of CCMV RNA3 are dispensable for individual systemic infection, these segments contribute to the competitive fitness of the virus and influence interaction with the host, as evidenced by symptom response.