Hepatitis B virions in plasma (Dane particles) are known to contain small circular DNA molecules. The experiments described here indicate that virions in plasma, as well as particles from hepatitis B virus-infected human liver, also contain viral DNA-RNA hybrid molecules, and deoxynucleotides can be incorporated into the DNA of these hybrids by DNA polymerase activities in the virions. Thus, two viral DNA synthetic reactions appear to take place in virions: repair of the single-stranded region of circular DNA molecules and synthesis or elongation of the DNA strand of DNA-RNA hybrid molecules. Centrifugation of virion nucleic acid to equilibrium in Cs2SO4 density gradients revealed the presence of viral DNA-RNA hybrid molecules over a density range of 1.45 to 1.60 g/cm3. Distinct species of hybrid molecules were found with an average density of 1.57 g/cm3 in Dane particles and 1.52 and 1.57 g/cm3 in particles from liver. Fractionation of nucleic acid from Cs2SO4 density gradients by gel electrophoresis demonstrated that the majority of hybrid molecules migrated faster than molecules with the density of pure DNA (1.42 g/cm3). One notable exception was the finding of DNA-RNA hybrid molecules migrating slower than open circular viral DNA. Characterization of viral DNA-RNA hybrids by heat denaturation Cs2SO4 density gradient fractionation, and recombinant M13-HBV single-stranded probe hybridization revealed that the hybrid molecules consisted of viral plus-strand RNA hydrogen bonded to viral minus-strand DNA sequences. Data obtained by pancreatic ribonuclease digestion revealed that the hybrid molecules at density 1.45 to 1.52 g/cm3 contained HBV RNA strands base paired over only part of their length in contrast to the hybrid species at density 1.57 g/cm3 which contained RNA strands apparently base paired over most of their length. Further characterization showed that the hybrid at 1.57 g/cm3 contained genome-length minus-strand viral DNA. The experiments rule out the possibility that the hybrid molecules are transcriptional complexes. Data presented in a companion manuscript indicate that the hybrid molecules may represent intermediates in the synthesis of viral DNA in the endogenous DNA polymerase reaction.