Abstract Isothermal titration calorimetry (ITC) was used to measure the binding affinity and thermodynamics of a cocaine-binding aptamer as a function of pH and NaCl concentration. Tightest binding was achieved at a pH value of 7.4 and under conditions of no added NaCl. These data indicate that ionic interactions occur in the ligand binding mechanism. ITC was also used to measure the binding thermodynamics of a variety of sequence variants of the cocaine-binding aptamer that analyzed which regions and nucleotides of the aptamer are important for maintaining high-affinity binding. Individually, each of the three stems can be shortened, resulting in a reduced binding affinity. If all three stems are shortened, no binding occurs. If all three of the stems in the aptamer are lengthened by five base pairs ligand affinity increases. Changes in nucleotide identity at the three-way junction all decrease the affinity of the aptamer to cocaine. The greatest decrease in affinity results from changes that disrupt the GA base pairs and the identity of T19.