RNA aptamers with 2'-fluoro-pyrimidine chemistry were previously selected by in vitro evolution to bind to monomeric HIV-1 gp120 from the R5 strain BaL. A group of 36 novel aptamers were cloned and sequenced from the heterogeneous pool and were tested for their ability to bind to gp120. The diversity of the RNA secondary structure of these, and 27 aptamers isolated previously, was analysed using a bioinformatics approach. This showed that eight aptamers contain a common branched motif, and RNA mutagenesis indicated that this structure is probably required for gp120 binding. Chemically synthesised derivatives of one such aptamer, B40, were designed and tested for binding to gp120. Truncation was found to decrease their binding, but the introduction of point mutations to stabilise the branched conformation and 2'-O-dimethylallyl-modified residues to stabilise helices increased binding to levels greater than that of the parental aptamer. The aptamer epitope on gp120 was mapped by testing aptamer binding to alanine-scanning mutants and deletion mutants of gp120 using a novel plate-based assay. This study showed that the aptamer binding site overlaps with the CCR5 epitope and is confined to four key residues at the base of the V3 loop, one of which is highly conserved. This finding may account for the observation that a number of aptamers were shown previously to neutralise a range of HIV-1 R5 clinical isolates in PBMC cultures. Interestingly however, the aptamer was unable to neutralise HIV-1 pseudovirus in a cell line, which is most likely due to the increased levels of cell-surface CCR5 in cell lines compared to PBMC. Future work should focus on identifying the structure and epitopes of other anti-gp120 aptamers as well as testing neutralisation of HIV-1, HIV-2 and SIV by the B40-derived aptamers. These aptamers can be used as tools to investigate the HIV-1 entry pathway and also have the potential to be developed as anti-HIV-1 microbicides.