During transcription in vitro catalysed by the virion RNA polymerase of Aspergillus foetidus virus AfV-S in the presence of tritiated UTP, the virus double-stranded RNA becomes labelled in one strand, which has the same sequence as the single-stranded RNA transcripts produced. Most of the label incorporated into double-stranded RNA could be chased into single stranded RNA by further reaction with excess unlabelled nucleoside triphosphates. In reactions containing tritiated UTP the single-stranded RNA transcripts released after the first round of transcription were unlabelled. It is concluded that transcription in virions of AfV-S occurs by displacement of one of the strands of double-stranded RNA by the RNA strand being newly synthesised i.e. the reaction is semi-conservative with respect to double-stranded RNA.