Nuclear viral RNAs synthesized early in productive infection with adenovirus 2 were analyzed by hybridization to specific viral DNA fragments. Radioactive RNAs were subjected to electrophoresis in polyacrylamide gels containing 98% formamide, and the fractionated RNA was hybridized to specific DNA fragments generated by cleavage with endo R-Eco Ri or endo R-Sma l. The viral genes expressed early in infection are located in four different segments of the genome. When nuclear RNA was hybridized to DNA fragments representing each of these segments, discrete RNA size classes were detected. For each of these four regions of the genome, some of the discrete nuclear RNAs were larger than the cytoplasmic mRNAs. As calculated from electrophoretic mobility, the molecular weight of these nuclear RNA size classes ranged from 15% to several fold greater than that of the corresponding cytoplasmic mRNAs. Hybridization-inhibition experiments were performed to analyze the sequences present in one of these larger nuclear transcripts, a 22S RNA transcribed from the left-hand end of the genome. This 22S nuclear RNA was transcribed from the same strand as the viral mRNA, for it contained cytoplasmic sequences; it also contained sequences restricted to the nucleus. The larger nuclear RNAs may serve as precursors to the cytoplasmic viral mRNAs synthesized early in infection.