Primer-template-based double-stranded nucleic acids capable of binding human immunodeficiency virus reverse transcriptase (HIV-RT) with high affinity were used as starting material to develop small single-stranded loop-back DNA aptamers. The original primer-templates were selected using a SELEX (Systematic Evolution of Ligands by EXponential enrichment) approach and consisted of 46- and 50-nt primer and template strands, respectively. The major determinant of the approximately 10-fold tighter binding in selected sequences relative to control primer-templates was a run of 6.8 G residues at the 3' primer end. Sixty, thirty-seven, twenty-seven, and twenty-two nucleotide loop-back single-stranded versions that retained the base pairs near the 3' primer terminus were constructed. Both the 60- and 37-nt versions retained high affinity for RT with K(d) values of approximately 0.44 nM and 0.66 nM, respectively. Random sequence primer-templates of the same length had K(d)s of approximately 20 nM and approximately 161 nM. The shorter 27- and 22-nt aptamers bound with reduced affinity. Several modifications of the 37-nt aptamer were also tested including changes to the terminal 3' G nucleotide and internal bases in the G run, replacement of specific nucleotides with phosphothioates, and alterations to the 5' overhang. Optimal binding required a 4- to 5-nt overhang, and internal changes within the G run had a pronounced negative effect on binding. Phosphothioate nucleotides or the presence of a 3' dideoxy G residue did not alter affinity. The 37-nt aptamer was a potent inhibitor of HIV-RT in vitro and functioned by blocking binding of other primer-templates.