The human papovaviruses BK virus (BKV) and JC virus (JCV) both encode a large-tumor (T) antigen which are highly homologous to each other as well as to simian virus 40 (SV40) large-T antigen. This high conservation of amino acid sequence has resulted in overlapping antigenicity such that immunological differentiation of the large-T antigens from the three different viruses has been difficult. Here we describe the generation and characterization of a new monoclonal antibody (BK-T.1) which is specific for BKV large-T antigen and which does not cross-react with the other papovaviral T antigens. We show that when BK-T.1 is used in conjunction with preexisting monoclonal antibodies against SV40 large-T antigen, it is possible to identify the origin of papovavirus T antigens.