Early polyoma virus-specific RNA, in nuclei and cytoplasm of cells labeled with [(3)H]uridine, was analyzed by hybridization with filter-bound Hpa II fragments of polyoma DNA. About 40% of labeled cytoplasmic virus-specific RNA hybridized with Hpa II fragment 2, which represents about 40% of the region coding for E-strand mRNA's; less than 5% hybridized with fragments 1 or 3, which lie outside this region. A somewhat lower proportion (about 30%) of labeled nuclear virus-specific RNA hybridized with fragment 2, and a small but significant fraction (7 to 14%) hybridized with fragments 1 and 3. About two-thirds of the nuclear RNA which hybridized to fragment 1 was complementary to the E strand, and one-third was complementary to the L strand. Results did not vary greatly in samples labeled for periods of from 15 min to 3 h. The major species of pulse-labeled nuclear polyoma-specific RNA sedimented at 22S and thus is slightly larger than the 19S cytoplasmic mRNA. These results show that most early nuclear RNA ( approximately 75%) is transcribed from the region of the E strand, which codes for early mRNA's, and that there is probably a site at which transcription is terminated at the end of this region. However, a small amount of early nuclear RNA ( approximately 15%) is transcribed from the remainder of the E strand, perhaps by readthrough of this termination signal. In addition, there is a small amount of transcription from the L strand, whose significance is unclear. Neither the L-strand transcripts nor the nonmessenger E-strand transcripts are transported to the cytoplasm.