The results of potential of mean force (PMF) calculations for the distinct stages of proton conduction through the gramicidin A channel, including proton migration, reorientation of the water file and negative ion defect migration, are presented. The negative ion defect migration mechanism was hypothesized in experimental studies but was not considered previously in molecular dynamics simulations. The model system consisted of the peptide chains constructed on the base of the structure PDBID:1JNO, the inner file of nine water molecules and external clusters of water molecules placed at both ends of the channel. Potential energy functions were computed with the CHARMM/PM6/TIP3P parameters. The results obtained for proton migration and water file reorientation are basically consistent with those reported previously by Pómès and Roux (Biophys J 82:2304, 2002) within the similar approach. For the newly considered mechanism of negative ion defect migration from the channel center to the end of the water file we obtain the energy 3.8 kcal mol(-1) which is not considerably different from the activation energy of water reorientation, 5.4 kcal mol(-1). Therefore this mechanism may principally compete for the rate-limiting step in proton conduction in gramicidin.