Intact cores of adenovirus type 5 were transcribed in vitro in extracts derived from uninfected HeLa cells, and the products were analyzed by hybridization to restricted viral DNA. Predominant transcripts from cores were distinctly different from transcripts generated from deproteinized DNA. The extracts contained RNA polymerases II and III. Transcription from cores was completely inhibited by low levels of alpha-amanitin, and there was little or no hybridization of products to the region of the genome encoding the small polymerase III-dependent virus-associated RNA that was a major transcript from the DNA template. The major transcripts from cores hybridized to the rightward-transcribed strand of the region between 75 and 89 map units, early region 3 of the adenovirus genome. There was several-fold more transcription of this region in cores than of the region downstream from the major late promoter, which predominates among polymerase II transcripts from DNA.