AbstractAn assay based on nucleic acid hybridization detects and quantitates hepatitis B virus (HBV) DNA in particles present in serum. This assay allows rapid examination of multiple samples and is sensitive and reproducible; serum samples are treated with proteolytic enzyme and detergent and then extracted with phenol and chloroform. The deproteinized extracts which may contain HBV DNA are made alkaline to denature the DNA, neutralized, and bound to nitrocellulose filters in 3‐mm in diameter “spots” in a special 96‐well filtration apparatus. HBV DNA is detected by its ability to hybridize with32P‐labeled DNA prepared from recombinant plasmids containing the complete HBV genome. After hybridization, the nitrocellulose is washed and autoradiographed; samples containing HBV produce spots on X‐ray film whose intensity (in the linear exposure range of the film) is proportional to the amount of HBV DNA in the serum sample. The assay is specific and sensitive, correlates with infectivity of sera titered in chimpanzees as well as biophysical parameters, and is in agreement with serological indicators of H