Small circular single-stranded DNA satellites, termed DNAbeta, have recently been found associated with some geminivirus infections. The DNA beta associated with Cotton leaf curl virus is responsible for symptom expression of a devastating disease in Pakistan. Mutagenesis of DNA beta revealed that the complementary-sense open reading frame (ORF) betaC1 is required for inducing disease symptoms in Nicotiana tabacum. An ORF present on the virion-sense strand betaV1 appeared to have no role in pathogenesis. Tobacco plants transformed with a betaC1 ORF under the control of the Cauliflower mosaic virus 35S promoter or with a dimeric DNA beta exhibited severe disease-like phenotypes, while plants transformed with a mutated version of betaC1 appeared normal. Northern blot analysis of RNA from the transgenic plants, using strand-specific probes, identified a single complementary-sense transcript. The transcript carries the full betaC1 ORF encoding a 118-amino acid product. It maps to the DNA beta at nucleotide position 186 to 563 and contains a polyadenylation signal 18 nt upstream of the stop codon. A TATA box is located 43 nt upstream of the start codon. Our results indicate that betaC1 protein is responsible for DNA beta-induced disease symptoms.