To obtain information about cytoplasmic structures and functions involving the entry of simian virus 40 virions into cells, we examined whether the inhibitors that affect the functions and/or structure of lysosomes, cell membrane, and cytoskeletons inhibit expression of nuclear T antigen in the SV40-inoculated rat 3Y1 and monkey CV-1 cells. Chloroquine, methylamine, and butylamine did not inhibit T-antigen expression, suggesting that lysosomal acidification is not required for establishment of infection. Cytochalasin B had no effect, suggesting that microfilaments are not involved. Monensin, colcemid, and amantadine each inhibited T-antigen expression at doses causing no obvious cytotoxicity. Maximal inhibition was seen when these inhibitors were added to the cultures within 1 hr (monensin), within 4 hr (colcemid), or within 12 hr (amantadine) after virion adsorption to the cell surface. When the inhibitor was present in the virus-inoculated cultures for 24 hr and then removed, nuclear T antigen began to be expressed at 4 hr (monensin), 9 hr (colcemid), or 1 hr (amantadine) after removal of the inhibitors. Results of SDS-PAGE analysis of immunoprecipitated radiolabeled proteins of infected cells revealed that amantadine inhibited synthesis of large and small T antigens as well as general protein synthesis. Inhibition by colcemid may be due to disruption of microtubules, because other microtubule-disrupting agents (colchicine, vinblastine, nocodazole, and podophyllotoxin) also inhibited appearance of nuclear T antigen but lumicolchicine and taxol did not. Electron microscopy revealed that, in the presence of colcemid, although the adsorbed virions were readily internalized to form pinosomes, vectorial movement of the pinosomes to the nucleus appeared to be inhibited. Results of electron microscopy also suggest that inhibition by monensin may occur mainly in internalization of adsorbed virions and that the inhibition is leaky such that the early steps of infection proceed slowly in the presence of monensin. We conclude that monensin, colcemid, and amantadine interfere with mutually different early events of SV40 infection.