Transcription of the genomes of the defective parvovirus, adenovirus-associated virus (AAV) type 2, and a helper virus, adenovirus (Ad) type 2, was studied in nuclei isolated from infected KB cells. As measured by the incorporation of [ 3H]UTP into acid-insoluble product, nuclei prepared 15.5 hr after coinfection with AAV and Ad (AAV-Ad nuclei) synthesized RNA for 10 min. Total RNA synthesis in AAV-Ad nuclei was greater than in uninfected nuclei but was only about 1 2 that observed in nuclei from cells infected with Ad alone (Ad nuclei). DNA-RNA hybridization with RNA samples from the AAV-Ad nuclei revealed that the in vitro synthesis of AAV-RNA (14% of total) was twice that of Ad-RNA (6% of total). When Ad or AAV-Ad nuclei were assayed at various times after infection, total RNA production was comparable until AAV transcription was well established (12 hr); thereafter, a relative depression of total RNA synthesis was observed in the AAV-Ad nuclei. However, from 12 hr on, more AAV-RNA than Ad-RNA was synthesized in AAV-Ad nuclei, and a comparison with Ad nuclei suggested a preferential inhibition of Ad-RNA transcription in the AAV-Ad nuclei. Finally, it was found that AAV, Ad, and total RNA synthesis shared the same optimum salt concentration [0.02 M (NH 4) 2SO 4], and that 95% of Ad-RNA synthesis and greater than 99% of AAV-RNA synthesis were inhibited by levels of α-amanitin which blocked cellular RNA polymerase II activity. These data suggest that the AAV genome is also transcribed by the same polymerase that transcribes the bulk of Ad-RNA and cellular heterogeneous nuclear RNA.