Initially, we investigated the distribution of chemicals in dried wood samples using a standard TOF-SIMS and obtained two-dimensional images of chemical distribution in various wood samples. However, during drying as part of the sample preparation, some artifacts could occur because water-soluble chemicals could be moved, drained, or changed at that time. To understand the function of chemicals in living trees, it is important to reveal the distribution of chemicals in planta. For this purpose, we developed the new cryo-TOF-SIMS/SEM system to analyze chemical distribution in frozen-hydrated samples. This new system consists of a glove box, a cryo-SEM, and a cryo-TOF-SIMS, which are interconnected by a cryo-vacuum shuttle. This system was then applied to freeze-fixed Cryptomeria japonica xylem. Potassium, ferruginol, a major wood chemical of C. japonica, and water were simultaneously detected in frozen-hydrated wood tissues. For accurate detection, the sublimation of ice from sample surfaces must be prevented. After the TOF-SIMS analysis of the surface, the sample was cut to make the next sample surface in the glove box and cryo-TOF-SIMS analysis was performed again. Continuing with this procedure, we obtained a three-dimensional analysis data of TOF-SIMS images. The maximum height was 1000 µm. These results suggest that this new system is useful for revealing the chemical distribution of wood tissue in planta. Copyright © 2012 John Wiley & Sons, Ltd.