Four virus-specific transcripts have been identified in murine cells infected with Minute-Virus-of-Mice (MVM). These RNAs, 4.8, 3.3, 3.0 and 1.8 kilobases in length, designated R1 to R4 respectively, are all transcribed from the virion (-) strand of DNA and they are all polyadenylated and spliced. The R1 transcript is derived from sequences that reside on the genome between 4.0 and 95 map units (mu). Transcript R2 is composed of exon sequences derived from mu coordinates 4.0-10.0, 40-46 and 48-95. The most abundant RNA, R3, is transcribed from sequences mapping between 40 and 95 mu. All three of these RNAs have a short intron sequence between 46-48 mu removed. The least abundant transcript, R4, has not been mapped precisely, however it hybridizes with all three EcoRI fragments which span the entire 5 kb genome. In vitro transcription of cloned restriction fragments of MVM DNA confirm the existence of functional promoters at map coordinates 4.0 and 39 and sequence analysis of these regions of the viral DNA reveal the characteristic features of RNA polymerase II promoters. These results indicate that MVM DNA encodes two overlapping transcription units with separate promoters near the left end (4.0 mu) and middle (39 mu) of the genome.