The nonanucleotide motif TAATATTAC occurs in the intergenic region of all geminiviruses that have been examined to date. The motif is invariably located within the loop of a potential stem-loop structure that has been implicated in viral DNA replication. To investigate the contribution of these sequences to virus proliferation, African cassava mosaic virus (ACMV) DNA B mutants have been screened for their ability to infect Nicotiana benthamiana when co-inoculated with DNA A. Mutants in which the putative stem structure was altered by the introduction of single nucleotide mismatches remained as infectious as the wild-type virus and the mutations were retained in the progeny. Mutants containing nucleotide substitutions within the loop sequences were similarly infectious but analysis of progeny showed that in most cases wild-type sequences were restored by recombination with DNA A. Stem-loop deletion mutants of both genomic components were not infectious when co-inoculated, although they were once again efficiently rescued by recombination when inoculated with the wild-type components. Co-inoculation of genomic components containing the motif TAGTATTAC did not result in a systemic infection while mutants containing the motif TAATACTAC were infectious and the mutation was stable. The results demonstrate that ACMV will tolerate some modification to this highly conserved region of the genome that might allow more precise mapping of the position at which the viral DNA is nicked during replication.