This study demonstrates that populations of defective interfering Semliki Forest virus (DI SEV) are heterogeneous particularly in respect of their interference properties. Interference was quantified by two assays, one measuring inhibition of the yield of infectious progeny virus, and the other measuring reduction in virus-directed RNA synthesis; for 11 different DI SFV preparations a ratio of the two interference titres was calculated. These ratios varied up to 46-fold indicating that each DI virus preparation contained an interference activity that varied independently of the other. However, sister stocks made from the same parental inoculum had similar properties. The effects of different DI virus preparations on other parameters (virus polypeptide synthesis, yield of DI virus and yield of infectious virus) were investigated using inocula with interference titres standardized by either assay. Co-inoculation of L929 cells with 50 p.f.u. SFV showed that these parameters varied independently of each other and of the DI virus inoculum. There was no correlation with the number of undiluted passages each DI stock had received. Direct evidence of physical heterogeneity was demonstrated by metrizamide density gradient centrifugation. Although infecting virus sedimented as a narrow band, DI SFV was distributed over a broad region of the gradient. Its position on the gradient indicated that DI SFV has a higher nucleic acid: protein ratio than standard virus. DI virus progeny obtained by using fractions of the gradient as inoculum were as heterogeneous as the unfractionated parent, confirming that DI viruses retain heterogeneity on passage.