The in vitro effect of monensin, a linear polyether, on the infection of Vero cells by BK polyomavirus, was investigated. Data reported in this paper showed an inhibition of BK viral replication by monensin as monitored by immunofluorescence and molecular hybridization. The inhibition of the synthesis of viral nuclear T antigen and the lack of production of viral mRNAs in monensin-treated cells suggest that the effect of this ionophore takes place at the level of the viral DNA delivery, by blocking the uncoating of BK virus or its transport to the nucleus.