Antisense oligonucleotides are currently being used in numerous laboratories as potential anticancer and antiviral agents. The unique replication cycle of hepatitis B virus (HBV) contains several different steps which are potentially amenable to modulation by these molecules. We have examined the ability of 56 different single-stranded, oligodeoxyribonucleotides (14-23 nucleotides in length), which target several HBV-specific functions, to inhibit HBV replication in the human hepatoblastoma cell line, 2.2.15. None of the oligonucleotides examined were toxic at concentrations up to 500 microM. Oligonucleotides directed against the HBV surface antigen (HBsAg) gene (S gene), the preS1 open reading frame, and the HBV core antigen (HBcAg) gene (C gene) were effective at depressing virus production, while molecules targeting the HBV e antigen (HBeAg) open reading frame and the HBV polymerase (POL) gene were ineffective. Oligonucleotides directed against the HBV encapsidation signal/structure (epsilon) comprised some of the most effective antiviral molecules against HBV. None of 5 oligonucleotides complementary (i.e., 'sense' orientation) to the antisense oligonucleotides targeting HBsAg, HBcAg, HBeAg, preS1 and POL had any measurable effect on HBV production. The relative effectiveness of oligonucleotides targeting the S and C genes on HBV replication was highly correlated with an effect on HBsAg or HBcAg levels, respectively. None of the antisense oligonucleotides examined affected either the levels or the sizes of HBV-specific RNA transcripts. Since antisense oligonucleotides can exert biologic effects on HBV in 2.2.15 cell cultures in a sequence-specific manner which are consistent with predicted modes of action, such molecules may have practical applications in the therapy of chronic HBV infection.