Recent advances in statistical mechanical theory can be used to solve a fundamental problem in experimental thermodynamics. In 1997, Jarzynski proved an equality relating the irreversible work to the equilibrium free energy difference, AG. This remarkable theoretical result states that it is possible to obtain equilibrium thermodynamic parameters from processes carried out arbitrarily far from equilibrium. We test Jarzynski's equality by mechanically stretching a single molecule of RNA reversibly and irreversibly between two conformations. Application of this equality to the irreversible work trajectories recovers the ΔG profile of the stretching process to within kBT/2 (half the thermal energy) of its best independent estimate, the mean work of reversible stretching. The implementation and test of Jarzynski's equality provides the first example of its use as a bridge between the statistical mechanics of equilibrium and nonequilibrium systems. This work also extends the thermodynamic analysis of single molecule manipulation data beyond the context of equilibrium experiments.