The pathogenic human parvovirus B19 contains a promoter at map unit 6 (B19p6) of the viral genome, expression from which is largely restricted to human cells in the erythroid lineage, whereas a putative promoter at map unit 44 (B19p44) is inactive during a natural viral infection. Although nonerythroid human cells, such as HeLa and KB, allow expression from the B19p6 promoter but not from the B19p44 promoter following DNA-mediated transfection, little expression from the B19p6 promoter occurs following recombinant virus infection (S. Ponnazhagan, X.-S. Wang, M.J. Woody, F. Luo, L.Y. Kang, M.L. Nallari, N.C. Munshi, S.Z. Zhou, and A. Srivastava, submitted for publication). However, significant expression from the B19p6 promoter as well as the B19p44 promoter could be detected in a human 293 cells line that expresses the adenovirus early gene products, suggesting that coinfection with adenovirus might mediate transcriptional transactivation of the B19 promoters in nonpermissive cells. Expression of the firefly luciferase reporter gene from the B19 promoters was evaluated either following plasmid transfection or following infection with the recombinant adeno-associated virus type 2 vectors. Both B19p6 and B19p44 promoters could be transactivated by coinfection with adenovirus in nonpermissive human cells, although the extent of transactivation of the B19p44 promoter was significantly lower than that of the B19p6 promoter. Expression of the adenovirus E1A proteins was necessary and sufficient for the observed transactivation of the B19 promoters. These studies further illustrate that the underlying molecular mechanisms of transactivation of parvovirus promoters in general by the adenovirus early proteins have similarities with those of the well-documented transactivation of the adeno-associated virus type 2 promoters.