The amount of topoisomerase I specific mRNA increases three- to fivefold during the early phase of infection of HeLa cells with adenovirus-5. The observed increase in specific mRNA is mainly due to an increased rate of transcription of the gene. In human 293 cells, which constitutively express the viral E1A and E1B genes, we determined an elevated level of topoisomerase I mRNA, comparable to the amount of mRNA present in HeLa cells early after infection with adenovirus. In contrasts, in HeLa cells infected with adenovirus dI312, a mutant were the E1A region had been deleted, the amount of topoisomerase I mRNA remained constant, unless the cells were superinfected with wild type virus. Our experiments indicate that the topoisomerase I gene is transactivated by an early adenovirus protein product coded by the E1A region. In contrast to the increase in mRNA synthesis, the amount of topoisomerase I protein and the topoisomerase I activity remain constant up to 24 hours after infection.