The method of chemical depurination is specific and quantitative for releasing pyrimidine oligonucleotides from DNA and should prove useful in a general procedure for sequencing DNA. In this report, the fractionation of 32P-labelled pyrimidine oligonucleotides from depurinated fd DNA on a two-dimensional thin-layer system is described. This system resolves the depurination products according to size and base composition. In fd DNA there are eight large polypyrimidine nucleotides of nine base residues or greater and most of these nucleotides are present in single molar yield. A technique employing partial digestions with spleen and snake venom phosphodiesterase, and fractionation of the partially degraded products with the two-dimensional thin-layer system, has been developed for sequencing these large pyrimidine oligonucleotides. The largest pyrimidine oligonucleotide in fd DNA contains 20 residues and has a sequence of 5′-C-T-T-T-C-T-T-C-C-C-T-T-C-C-T-T-T-C-T-C-3′. None of the oligonucleotides sequenced appears to be derived from the coat protein gene.