The effect of suramin on duck hepatitis B virus (DHBV) infection was investigated in vivo. Suramin pretreatment of Pekin ducklings completely prevented DHBV infection. In contrast, suramin given at the time of or after inoculation with DHBV did not inhibit viral infection, replication, or gene expression. These data indicate that suramin effectively blocks the early stages of DHBV infection in vivo.