Replication of hepatitis A virus (HAV) in the human hepatoma-derived PLC/PRF/5 cell line was neither inhibited in the presence of various concentrations of guanidine or D-2-(alpha-hydroxybenzyl)benzimidazole (D-HBB), nor were the two chemicals effective in combination. Under identical conditions, however, replication of poliovirus type 1 was inhibited. Tracer experiments with radiolabelled guanidine and D-HBB also furnished no evidence that the two antiviral substances were metabolized gradually to inactive derivatives in PLC/PRF/5 cells. Therefore, it is concluded that resistance to the action of guanidine and D-HBB is an inherent characteristic of HAV. However, the insensitivity of HAV to these drugs does not exclude the virus from the family of picornaviruses.