Jarzynski's equality provides a non-equilibrium method for calculation of free energies, which could have wide-ranging applications in complex molecules. Tests of Jarzynski's equality have mostly focused on simple systems or near-equilibrium processes. Here we present a benchmark test that compares the results of free energy calculations for a potassium ion in carbon nanotube and gramicidin channel, which can be identified with simple and complex systems. A good agreement is found between the free energy results obtained using Jarzynski's equality and established methods in the carbon nanotube, but discrepancies occur in gramicidin, indicating relaxation problems in application of Jarzynski's equality to complex biomolecular processes. (c) 2007 Published by Elsevier B.V.