The effect of hydrogen bonding on the diffusivities of two almost identical fluorescent probes through polyamides was examined using fluorescence recovery after photobleaching (FRAP). The two molecules have the same geometry and a negligible difference in their molecular weight, the main difference confined is one characteristic group able to form a hydrogen bond with the polyamide matrices. Moreover, the effect of hydration on the diffusion coefficients and the plasticization activation energy is also studied as the experiments are repeated in various relative humidity environments. The differences in the diffusivities of the two molecules due to hydrogen bonding were as much as one order of magnitude regardless the matrix or the water content. In addition, it is shown that the plasticization activation energy of diffusion is also affected by matrix-diffusant interactions as the relevant values of the two examined diffusing molecules showed noticeable differences due to hydrogen bonding. POLYM. ENG. SCI., 51:532-541, 2011. (C) 2010 Society of Plastics Engineers