Cultured hepatoma cells (HepG2) were cotransfected with two different plasmids carrying a head-to-tail dimer of recombinant hepatitis B virus (HBV) DNA cloned from deletion mutants isolated from the circulation of persistently infected hosts. They were tested for the secretion of viral particles with mutant genome encapsidation. A recombinant plasmid defective in the S gene and one defective in both the C and P genes complemented in trans for the production of viral particles. Mutant genomes from either of the recombinants were encapsidated. Similarly, a recombinant defective in the C gene and another defective in the P gene trans-complemented for the production of viral particles containing mutant genomes. A hepatoma cell line with integrated HBV DNA sequences defective in the C and P genes (PLC/PRF/5) when transfected with a recombinant defective in the S gene produced viral particles with the HBV genome from the transfecting recombinants. These results confirm the expected trans-complementation among the S, C and P genes of HBV, when either episomal or integrated into chromosomes, for the maintenance of defective HBV mutants in persistently infected hosts.