Exposure of vesicular stomatitis virus-infected Chinese hamster ovary cells to cycloheximide results in the complete transcription of virion ribonucleic acid (RNA) into only 28S and 13 to 15S viral-specific RNA species. These RNA are identical to viral messenger RNA by the following criteria: size, single-strandedness, complementarity to virion RNA, and formation of messenger ribonucleoproteins. This transcription represents the intracellular enzymatic activity of the virion-associated polymerase and is shown to be dependent on input multiplicity. Intracellular transcription differs from in vitro polymerase activity in having a temperature optimum of 34 to 37 C and in synthesizing 28S as well as 13 to 15S messenger RNA species. Addition of interfering quantities of defective T particles to these cycloheximide-treated cells, either an hour before or at the same time as standard B particles of vesicular stomatitis virus, does not alter the rate of transcription nor does it change the sucrose gradient pattern of the viral RNA species. From these results it is concluded that the RNA of defective T particles does not serve a transcriptive function and probably interferes through the replicative mechanism for virion RNA synthesis.