Broad bean mottle bromovirus (BBMV) was shown to accumulate RNA2-derived defective interfering (DI) RNAs [Romero et al., Virology 194, 576-584 (1993); Pogany et al., Virology 212, 574-586 (1995)]. In this work, we utilize three sets of BBMV RNA2-derived artificial DI RNA constructs to determine factors that affect the accumulation of defective RNAs in planta. One set of deletion constructs was used to localize sequences required for efficient accumulation within the 5' 883 nt and the 3' 387 nt of the DI RNAs. The second set had a gradually increasing size of 3' nested deletions to determine the minimal length required for efficient DI RNA accumulation. The smallest DI RNA still accumulating in plants was found to be 1712 nt long. The third set consisted of frameshift mutants which showed that at least 64.4% of BBMV DI RNA sequences must have the 5' portion of the 2a open reading frame to ensure efficient accumulation. The importance of these factors in the selection of DI RNAs is discussed.