Six restriction fragments of Ad2 DNA which contain sites for RNA initiation have been identified by their ability to hybridize nascent labeled RNA less than 1 kb in length. Four RNA initiation sites early in infection are identified in regions where previous work (Flint, 1977) had mapped mRNAs. The major late RNA initiation site is the origin of a giant nuclear transcript extending from approximately 16.3 map units to (or close to) 100 units at the end of the genome. This transcription unit encompasses at least four or five mRNA sites; processing of this long transcript appears necessary to generate the mRNA.