Abstract The DNA duplex corresponding to the entire length (77 nucleotides) of the major yeast alanine transfer RNA has been synthesized. The duplex [A](paper CXII), corresponding to nucleotide sequence 1 to 20 and containing a single-stranded end (nucleotides 17 to 20) was joined to the duplex [B](paper CXIII), corresponding to nucleotide sequence 17 to 50 and containing single-stranded ends (nucleotides 17 to 20 and 46 to 50). The resulting duplex [A-B], nucleotide sequence 1 to 50, was characterized. The duplex [B] was also joined to [C 5] (paper CXIV), which corresponds to nucleotide sequence 56 to 77 in the presence of the “bridge” decanucleotide d-G-G-A-G-C-G-C-G-C-T (nucleotides 46 to 55) to give the duplex [B–C](nucleotide sequence 17 to 77). The entire duplex [A-B-C] was prepared in two alternative ways. In the first, preformed [B + C] was joined to the duplex [A]. In the second, preformed duplex [A+B] was joined to either the duplex [C 6](paper CXIV) or the duplex [C 5] in the presence of the decanucleotide d-[5′- 32P]-d-G-G-A-G-C-G-C-G-C-T. The duplex [A-B-C] was characterized.