A new method for determining the 3'-terminal nucleotide of a DNA strand is presented. Use is made of the fact that one (and only one) 2',3'-dideoxyribonucleotide can be added to the 3'-end of a DNA fragment with calf thymus terminal transferase. Addition of more than one nucleotide analog per strand is impossible due to the absence of a 3'-terminal hydroxyl group. If the terminatind dideoxyribonucleotide contains an (alpha 32p) label, the resulting 3'-blocked strand can be digested by "nearest neighbor" techniques and the original 3'-endgroup determined. Picomole quantities of DNA strands can be labeled and the 3'-end determined.