Relatively few thermodynamic parameters are available for RNA triloops. Therefore, 24 stem-loop sequences containing naturally occurring triloops were optically melted, and the thermodynamic parameters ΔH°, ΔS°, ΔG°(37), and T(M) for each stem-loop were determined. These new experimental values, on average, are 0.5 kcal/mol different from the values predicted for these triloops using the model proposed by Mathews et al. [Mathews, D. H., Disney, M. D., Childs, J. L., Schroeder, S. J., Zuker, M., and Turner, D. H. (2004) Proc. Natl. Acad. Sci. U.S.A. 101, 7287-7292]. The data for the 24 triloops reported here were then combined with the data for five triloops that were published previously. A new model was derived to predict the free energy contribution of previously unmeasured triloops. The average absolute difference between the measured values and the values predicted using this proposed model is 0.3 kcal/mol. These new experimental data and updated predictive model allow for more accurate calculations of the free energy of RNA stem-loops containing triloops and, furthermore, should allow for improved prediction of secondary structure from sequence.