T4 polynucleotide kinase has been used to end-label specific RNA purified by multiple hybridizations to nitrocellusose-bound DNA. The pico models of ends of a specific mRNA transcribde from the chromosome, even from several liters of Escherichia coli, give concentrations perhaps 2000-fold below the K m value of kinase-RNA substrate. In such a reaction, optimal incorporation was observed with increasing ATP concentration to ⩾ μM (⩾ 15 mCi of carrier-free [ 32P]ATP in a 300–500 μl reaction). The unreacted ATP (> 150-fold excess) could best be eliminated by multiple gel filtrations rather than by precipitation, ion exchange chromatography or dialysis. The [5′- 32P]RNA was digested with T1 or pancreatic RNase and the [5′- 32P]olilgonucleotides separated by size in a 20% polyacrylamide gel. Olilgonucleotides of a specifc size were separated sufficiently by a second dimension electrophoresis on cellulose acetate. We have used partial alkali digestion is sequencing the purified oligonucleotides. As opposed to other digestions, alkali produces 5′-3′-diphospho-oligonucleotides whose mobilities can differ from those of the monophosphates e.g., much longer running times in conventional homochromatography.