The presence of serological markers of hepatitis B virus (HBV) infection and of hepatocellular HBV DNA were investigated in 19 HBsAg-negative patients with clinically and histologically significant chronic liver disease. Four cases negative for antibodies to HBsAg (anti-HBs), to the core antigen (anti-HBc), and to the e antigen (anti-HBe) were classified as non-A, non-B hepatitis. The remainder, positive for one or more of the three antibodies, were classified as hepatitis B. Histologic diagnosis was chronic active hepatitis in five, chronic persistent hepatitis in 11, micron-odular cirrhosis in two, and fatty liver in one patient. The DNA extracted from limited amounts of liver biopsies, without cleavage by restriction endonucleases, was analyzed by the Southern blot technique for the presence of episomal HBV DNA. Autoradiographs showed a single band of less than 4.0 kilobase (kb) corresponding to the monomeric form of HBV DNA in five patients, several bands of larger forms (4.0 to 18.0 kb) in three patients, both the monomeric and the larger forms in eight patients, and no HBV DNA in three patients. While HBV DNA was detected in the hepatocellular DNA of six patients who underwent splenectomy, hybridization was negative with the DNA extracted from their spleens. The episomal viral DNA larger than 4.0 kb may represent concatemeric forms or free oligomers which could not be distinguished from rearranged and/or integrated viral DNA in the limited analyses of the hepatocellular DNA hydrolyzed with HindIII or EcoRI. Our observations suggest the presence of HBV-like agents in the liver of serologically HBsAg-negative patients with chronic liver disease.