A cell culture system for the evaluation of compounds which inhibit HBV replication (Korba and Milman, Antiviral Res. 15:217, 1991) has been developed into a standardized assay. Toxicity of test compounds was assessed by the uptake of neutral red dye under culture and treatment conditions which were identical to those used for the antiviral assays. A total of 667 separate cultures of 2.2.15 cells were evaluated for this study. In 86 untreated cell cultures, representing 15 experiments over a 24-month period, the levels of extracellular HBV virion DNA and intracellular HBV DNA forms were found to vary by less than 2.5-fold overall. Virion DNA in serum and intracellular viral DNA replication intermediates [RI] are the two most reliable and commonly followed markers of hepadnavirus replication in patients and experimental animals. In these assays, levels of extracellular HBV virion DNA and intracellular HBV RI were well correlated in 2.2.15 cells. Less correlation was observed between the levels of HBV virion DNA and the 3.2-kb episomal HBV genomes present in the cells. A threshold level of 22-37 intracellular replicating HBV genomes appeared to be required before virions were detected in the culture medium. The activities of several 2'-substituted and 3'-substituted deoxynucleoside analogs against HBV replication were compared using this standardized assay. Dideoxycytosine [ddC] and dideoxyguanosine [ddG] were the most selective 2',3'-dideoxynucleosides against HBV in 2.2.15 cells. Substitution of fluorine at the 2' position abolished the antiviral activity of ddC, but enhanced the selective antiviral activities of dideoxythymidine and dideoxyuracil. Several 2'-fluorinated pyrimidine arabinosyl furanosides, reported to be potent (but toxic) inhibitors of hepadnaviruses in vivo, demonstrated relatively low selective antiviral activities in 2.2.15 cells. The current data base allows for validation of any given set of test evaluations through statistical analysis of both the positive and the negative treatment controls present in each experiment; thus, relevant comparisons of the selectivity of anti-HBV activities for different compounds examined in future experiments can be made.