Four avian influenza viruses have been generated, each containing a single extra defective RNA segment in addition to the eight standard segments. Three of the extra RNAs were derived from segment 1 and the fourth from segment 2. Chick embryo fibroblast cells were infected with each virus, and a wild-type virus. Virus RNA was quantified in extracts of virus-infected cells and in virus released by 10 hr postinfection using reverse transcription and by Northern blot analysis. In the case of two of the viruses the presence of the defective RNA did not markedly affect the accumulation of virus RNA within the infected cell, but significantly and selectively reduced the amount of the "parent" segment in released virus. This effect was reduced in a third virus. In a fourth virus, defective RNA was found to be present at a low-input multiplicity and results were varied. Mixed infections of one of the viruses with a closely related wild-type virus resulted in reduction of the corresponding vRNA segment of the nondefective virus. We conclude that assembly of influenza virus segments is not a purely random process.