The N-terminal 70 residue "J-domain" of the Escherichia coli DnaJ molecular chaperone is the defining and highly conserved feature of a large protein family. Based upon limited, yet significant, amino acid sequence homology to the J-domain, the DNA encoding the T/t common exon of the simian virus 40 (SV40), JC, or BK polyoma virus T antigen oncoproteins was used to construct J-domain replacement chimeras of the E. coli DnaJ chaperone. The virally encoded J-domains successfully substituted for the bacterial counterpart in vivo as shown by (i) complementation for viability at low and high temperature of a hypersensitive bacterial reporter strain, and (ii) the restoration of bacteriophage lambda plaque forming ability in the same strain. The amino acid change, H42Q, in the SV40 T/t and the JC virus T/t exon, which is positionally equivalent to the canonical dnaJ259 H33Q mutation within the E. coli J-domain, entirely abolished complementing activity. These results strongly suggest that the heretofore functionally undefined viral T/t common exon represents a bona fide J-domain that preserves critical features of the characteristic domain fold essential for J-domain interaction with the ATPase domain of the Hsp70 family. This finding has implications for the regulation of DNA tumor virus T antigens by molecular chaperones.