Several strains of the broad bean mottle virus (BBMV), an icosahedral tripartite plant RNA virus, which show distinct reactions on certain plant hosts have been described (K. M. Makkouk et al., Neth. J. Plant Pathol. 94, 195-212, 1988). Here we report defective interfering (DI) RNAs encapsidated in two BBMV strains from Morocco and Tunisia. While not effective in some plants, these DI RNAs exacerbated the severity of symptoms in others. The most dramatic, lethal effect of DI RNAs has been found on pea (Pisum sativum, cv. Rondo) seedlings. Sequence analysis has revealed that the DI RNAs were derived by single in-frame central deletions of 448 to 537 nt in the corresponding genomic RNA2 components. A comparison of the intensities of full-length RNA2 bands from DI molecule-containing and DI molecule-deficient virion RNA preparations revealed that the DI RNAs decreased the level of RNA2 components in total RNA preparations. The differences between corresponding virion RNAs were much smaller. This suggests an interference with RNA replication. In vitro assays and an analysis of the polyribosomal RNA fractions confirmed the translational activity of DI RNAs. This paper reports the first description of natural DI RNAs in tripartite isometric plant RNA viruses.