Defective DNA forms of the geminivirus ageratum yellow vein virus (AYVV) have been identified in naturally infected Ageratum conyzoides plants. Several examples of the defective DNA have been cloned from purified virus-specific supercoiled DNA and characterized by sequence analysis. All are approximately half the size of AYVV genomic DNA, and all contain intergenic region sequences and the 5' terminus of gene C1 as well as additional sequences that are unrelated to the viral genomic DNA. The chimeric nature of the defective DNA distinguishes it from previously characterized geminivirus defective and satellite DNAs. The defective DNA ameliorates disease symptoms and causes a significant delay in the accumulation of viral DNA during the early stage of infection when coinoculated with the AYW genomic DNA into Nicotiana benthamiana, suggesting a biological role as a defective interfering DNA.