The genomic DNA of chicken anemia virus (CAV) was cloned and sequenced from a Japanese isolate CAA82-2. The nucleotide sequence of CAA82-2 isolate was 98% identical with that of the European Cuxhaven-1 strain (Noteborn et al., J. Virol. 65, 3131-3139, 1991). Nine open reading frames (ORFs) consisting of more than 100 nucleotides were found, i.e., four ORFs (CA1-CA4) on the plus strand and five ORFs (CA1R-CA5R) on the minus strand. These ORFs with the exception of CA4 are conserved between the two CAV isolates. All of these ORFs were expressed in Escherichia coli as fusion proteins with beta-galactosidase. By Western blot analysis, the CA2 and CA3 fusion proteins were found to react with CAV-infected chicken sera. Rabbit hyperimmune sera against the CA1, CA2, and CA3 fusion proteins were produced and tested their reactivity to CAV-infected cells. Two viral proteins with the apparent size of 54 and 16 kDa reacted with the antibodies against CA1 and CA3 fusion proteins, respectively. The 16-kDa protein, CA3, was suggested to be a major immunogen on CAV infection.