Low levels of hepatitis B virus (HBV) replication and serum Dane particles have been commonly observed in chimpanzee chronic HBV carriers. To evaluate the possibility of extrahepatic sites of replication, DNA from various organs of a chimpanzee HBV carrier were evaluated by Southern blot analysis. With cloned, repurified HBV DNA of 3.2 kilobases (Kb) as a hybridization probe under stringent conditions, analysis of liver DNA revealed a diffuse hybridization pattern below 3.2 Kb and full-length double-stranded HBV genomes at 3.2 and 1.8 Kb, the latter representing the supercoiled (CCC) form found in the nucleus. No HBV DNA was found in pancreas, muscle, renal, or adrenal gland. Analysis of splenic DNA revealed diffuse hybridization below 3.2 Kb within the cytoplasmic subcellular fraction, and full-length HBV genomic forms in the nuclear fraction of splenic tissue. Use of (-) and (+) strand-specific HBV DNA and RNA probes demonstrated asymmetric viral replication within the spleen cytoplasm as previously demonstrated in liver. Northern blot analysis of total RNA from chimpanzee spleen and liver revealed HBV RNA sequences in both of these tissues, suggesting active viral gene expression and/or replication in chimpanzee spleen as well as in liver. Elucidation of the splenic cell type supporting viral propagation may serve as a basis for development of a tissue culture system to study molecular events of HBV replication.