Defective DNA of tobacco leaf curl virus (TLCV) was identified in TLCV-infected tobacco plants. The defective DNA was cloned and sequenced. The sequence showed it was about half the size of the TLCV DNA-A, and was derived from TLCV DNA-A by a large deletion. The defective DNA contained the intergenic region and part of the AC1 (Rep) gene of TLCV, and also novel open reading frames (ORFs). The immunotrapping tests showed the defective DNA was associated with geminate particles, suggesting it could be encapsidated in virus particles. It was transmitted, along with full-length DNA-A, to tobacco plants by grafting and whitefly (Bemisia tabaci).