Ligation of an adapter oligonucleotide to a single-stranded cDNA is central to many molecular biology techniques. Current single-stranded ligation approaches suffer from low efficiencies and are strongly inhibited by preexisting DNA secondary structure. We develop an approach for ligating low concentrations of single-stranded DNAs to a DNA adapter with near-quantitative efficiency, unaffected by secondary structure in the target DNA. This efficient DNA ligation reaction will facilitate development of robust procedures for quantifying small amounts of highly structured cDNAs and their RNA templates.