The major late promoter of adeovirus-2 is located at coordinate 16.45 and initiates synthesis of nuclear precursors that are processed into mRNAs which fall into five 3- co-terminal families, L1-L5. These mRNAs all share a common tripartite 5' leader with a capped terminus encoded at th RNA initiation site. We show that the coorindate 16.45 RNA initiation site is also an early promoter, and yields transcripts detectable as early as 1 hr post-infection, prior to DNA replication and the early-late switch at 6-8 hr. Polyadenylated cytoplasmic RNA from the first 3- co-terminal family, L1, is also produced from the earliest stages of infection. L1 mRNA accumulates in the cytoplasm in the presence of cycloheximide, which blocks DNA replication and the onset of the late phase. Early nuclear RNA contains the same capped 5' terminal RNAase T-1 undecanucleotide and promoter proximal oligonucleotides present in late transcripts. This implies that precisely the same transcription start site is utilized for early L1 mRNA synthesis as is used during the late stage for L1-L5 late mRNA synthesis. In contrast to the early apperance of L1 mRNA, neither L2 nor L3 mRNAs are detected until 5-6 hr post infection. We cnclude that a major event in the Ad-2 early-late switch is a novel form of control which activates L2-L5 mRNA production.