A new approach to creating fluorescent signaling aptamers using fluorescent nucleotide analogues is presented. The fluorescence quantum yield of nucleotide analogues such as 2-aminopurine strongly depends on base stacking interactions when incorporated into double or single stranded DNA. This property is used to generate a binding-specific fluorescence signal. Aptamers for human alpha-thrombin, immunoglobulin E, and platelet-derived growth factor B were modified with fluorescent nucleotide analogues in positions that undergo conformational changes. The resulting signaling aptamers show a specific, binding-induced increase in the fluorescence signal of up to 30-fold. Conformation-changing positions in these aptamers were identified by screening a set of modified aptamer sequences that each included a fluorescent nucleotide analogue at a different position. The positions for these modifications were estimated by modeling the aptamer secondary structure. It is likely that this approach to producing fluorescent signaling aptamers is of general use for protein-binding aptamers because of their "induced fit" binding mechanism.