We have identified two major and approximately ten minor poly(A)-containing RNA species in S. cerevisiae which arise from in vivo transcription of the yeast plasmid, known as 2mu circle. The two major species, which are 1325 and 1275 bases in length, are transcribed from the two unique halves of the plasmid and extend into the inverted repeat sequences which separate the unique regions. The map positions of the minor transcripts, which range in length from 350 to 2600 bases, indicate that except for a small region of the genome in which no transcription is observed, both strands of the entire 2mu circle genome are transcribed. We also present evidence demonstrating that RNA transcribed from 2mu circular DNA is used to program the synthesis of specific proteins in yeast: that is, yeast RNA complementary to 2mu circle DNA can be translated in vitro to produce specific polypeptides of substantial size. Finally, the pattern of transcription of 2mu circle suggests the possibility that messenger RNA species are derived by cleavage of larger transcripts, and in addition, that the intramolecular recombination of 2mu circle which occurs in yeast functions as a genetic switch to allow separate expression of two sets of genes on the 2mu circle genome.