A 5 bp insertion was introduced into the BstEII site at nucleotide 2815 in DNA of hepatitis B virus (HBV) and a mutant HBV genome was produced, which coded for envelope and core proteins, but not for DNA polymerase, due to a frameshift. Cultured hepatoma cells (HepG2) were simultaneously transfected with a plasmid harbouring a tandem dimer of the mutant HBV DNA and another plasmid harbouring a tandem dimer of DNA of woodchuck hepatitis virus or duck hepatitis B virus. The replication of mutant HBV DNA, incapable of encoding DNA polymerase, was accomplished by cotransfecting woodchuck hepatitis virus DNA, but not by duck hepatitis B virus DNA. These results indicated a trans-complementation of the C and P genes in mammalian hepadnaviruses beyond a species barrier.