The distribution of viral RNA molecules in HeLa cells infected with adenovirus type 5 (Ad5) was determined by in situ hybridization at the ultrastructural level at an intermediate stage of nuclear transformation, when viral DNA synthetic activities were maximal but progeny viruses were still sparse. Transcription sites of the viral DNA were localized by short pulse, high resolution autoradiography. Nascent viral RNA was found mainly within the nuclear compartment identified at the peripheral replicative zone, which is known to be the main replicative site of Ad5 viral genomes. Viral RNA molecules also were present, but to a markedly lesser extent, within the contiguous single-stranded (ss) DNA accumulation site, another intranuclear virus-induced structure in which some replication of viral genomes also occurs. Two other virus-induced nuclear structures contained viral RNA, the occasional exceptionally enlarged clusters of interchromatin granules and the compact rings, both DNA-free structures of unknown significance but which might play a role in the process of maturation of the Ad5 primary transcripts. Viral messenger RNA molecules were localized over the large areas of the cytoplasm which contain numerous ribosomes. Our analysis of the effects of various enzymatic pretreatments of the sections of infected cells on the revelation of nascent RNA by in situ hybridization is reviewed.