Aptamers are synthetic, single stranded nucleic acids that bind specifically to the target protein. Aptamers have many potential uses including therapeutic and diagnostic applications. Most aptamers are identified through standard SELEX (Systematic Evolution of Ligands by EXponential enrichment) procedures that include a starting pool of nucleic acids with a region of random nucleotides flanked by fixed sequences. A main disadvantage of the traditional SELEX is the potential for the fixed sequences to "bias" the selection by interacting with nucleotides in the random region of the oligonucleotide. I developed a novel primer-free SELEX method for isolating single strand 30 nt DNA aptamers from a random sequence pool for HIV reverse transcriptase (HIV-RT), in which selection occurs in the absence of any flanking, fixed nucleotides. Selected aptamers bound ~10-20 fold tighter than starting material to HIV-RT. The selected aptamer (PF1) contained a motif of four diguanosine repeats. PF1 was compared to two classes of HIV-RT aptamers: a G-quadruplex aptamer (R1T family) from Michalowski et al., Nuc. Acids Res. 2008, and a primer-template aptamer developed by our lab (37 NT SELEX, DeStefano and Nair, Oligonucleotides 2008). PF1 was an effective inhibitor of HIV-RT in vitro and bound to RT about as tightly as a full primer-template. Both the R1T and 37NT SELEX were more potent HIV-RT inhibitors. This may be due in part to their large size. Circular dichroism spectroscopy indicated that PF1 does not form a G-quadruplex, while the R1T, consistent with previous results, is a parallel G-quadruplex. The effect of the primer-template aptamer on HIV inhibition in cell culture was also studied. Low micromolar concentrations of aptamer in the absence of a transfection agent inhibited replication in Jurkat cells without significant cellular toxicity. However constructs with similar structure also inhibit HIV replication leading us to conclude that aptamer blockage of replication may be due to alteration of cellular pathways. In addition to the possible contribution in developing novel nucleic acid-based HIV inhibitors, aptamer selection experiments can also shed light on how RT interacts with nucleic acids both in vitro and in cellular models.