Monolayer cell cultures of primary woodchuck hepatocytes, prepared by perfusing the liver in situ with collagenase type I, yielded hepatocytes with a viability of greater than 90% which could be held in culture for up to 3 months. Cultures of primary woodchuck hepatocytes were infected one day after plating with hepatitis delta virus (HDV) which had been passaged five times in woodchucks and was therefore identified as woodchuck hepatitis delta virus (WHDV). Replication of WHDV was demonstrated by the appearance of genomic WHDV RNA of ca. 1.7 kb beginning 7 days after infection, with an increase of copy numbers up to 2 weeks after inoculation. Synthesis of hepatitis delta virus-associated antigen (HDAg) in hepatocytes was detected by immunofluorescence staining of hepatocytes. Preincubation of the inoculum with rabbit sera containing antibodies against woodchuck hepatitis virus surface antigen (anti-WHs) reduced the infectivity of WHDV to an undetectable level compared with inocula which were treated with anti-WHs negative sera.