Thirty six cloned hybridomas have been isolated which produce monoclonal antibodies directed against simian virus 40 (SV40) large T tumour antigen. They have been shown to recognize at least six different epitopes along the T antigen polypeptide according to their reaction with the various truncated forms of T antigen expressed by adenovirus‐SV 40 hybrid viruses. Sixteen antibodies cross‐react with cells infected by the closely related human BK virus. Only two antibodies, PAb1604 and PAb1614, directed against different epitopes of the SV40 T antigen, cross‐react with polyoma large T tumour antigen which has a more limited amino acid sequence homology. This cross‐reaction is rarely seen with polyclonal antibodies. Monoclonal antibody PAb1620 gave nuclear immunofluorescence only with murine cells transformed by SV40 and was found to react with a complex of T‐antigen and 53 000‐dalton host‐coded protein. All the monoclonal antibodies react with nuclear T antigen and all but four antibodies stained the surface of SV40‐transformed cells. These were four of the five antibodies directed against the central third of the T antigen. Thus the monoclonal antibodies show that cell surface T antigen differs from nuclear T antigen, either in accessibility or structure.