Many naturally occurring RNA structures contain single mismatches. However, the algorithms currently used to predict structure from sequence are based on a minimal set of data which requires several approximations and assumptions to predict the stability of RNA duplexes containing single mismatches. Relative frequency and sequence patterns of single mismatches in naturally occurring RNA secondary structures were determined using a database containing 955 published secondary structures. Optical melting experiments were used to derive thermodynamic parameters for duplex formation for 28 oligoribonucleotides containing single mismatches. This data was combined with previously published data resulting in experimental data for the 30 most frequently occurring single mismatches in the database. Using this data, thermodynamic parameters specific for each type of loop have been derived, along with an AU/GU nearest neighbor parameter, in order to predict the thermodynamic contribution of unmeasured 1x1 loops. These new parameters, in conjunction with the experimental thermodynamics for frequently occurring single mismatches, will allow for a more accurate prediction of the free energy of RNA duplexes containing single mismatches and, furthermore, allow for improved prediction of secondary structure from sequence.