The RNA species encoded by the five early regions of adenovirus serotype 2 (Ad2) were isolated from the cytoplasm of HeLa cells at various times after infection either in the absence or in the presence of cycloheximide or cytosine arabinoside. The transcripts were characterized by electron microscopy of heteroduplexes formed with single-stranded Ad2 DNA. Each early region gave rise to a family of composite RNA species with up to four conserved segments spliced together after the deletion of intervening sequences. Transcripts from each family contained some sequences in common but differed in the lengths and positions of the conserved segments and thus had alternative splicing patterns. They were mapped on the adenovirus genome and the relative abundance of each species was determined. The map co-ordinates of the early region promoters were refined, based upon the indication that the 5′ ends of mature cytoplasmic RNAs are derived from promoter-proximal DNA sequences. The 5′ leader segment of the region 2 message for the single-stranded DNA binding protein changed during the course of infection, suggesting the utilization of different promoters at early and late times. The transition was prevented by blocking protein synthesis with cycloheximide. Transcription of region 3 was under the control of its own promoter at early times but could also be directed by the major r-strand late promoter at intermediate to late times. The internal splicing patterns of the region 3 transcripts seen at early times persisted at late times, but some molecules had the 5′ tripartite leaders common to late r-strand mRNAs. One of the major early region 3 transcripts contained three RNA segments which are also present in a presumptive processing intermediate for the fiber RNA, a late message which is separated from its promoter by early region 3. These extra segments in some fiber RNAs apparently reflect the recognition of early RNA splicing signals at late times. From the large, complex arrays of composite RNA structures, numerous insights into the RNA splicing mechanisms were inferred. © 1979.