As reactive oxygen species are important for many fundamental biological processes in plants, specific and sensitive techniques for their detection in vivo are essential. In particular, the analysis of hydroxyl radical (OH•) formation in biological reactions has rarely been attempted. Here, it is shown that spin trapping electron paramagnetic resonance (EPR) spectroscopy allows the detection and quantitative estimation of OH• production in vivo in one single cucumber seedling root. It is possible to localize the OH• production site to the growth zone of the root by varying the position of the intact seedling inside the resonator cavity of the EPR spectrometer. Moreover, the demonstration of impaired OH• formation in the root of the Arabidopsis mutant rhd2 impaired in a superoxide-producing Nicotimamide adenine dinucleotide phosphate (NADPH) oxidase has been accomplished. Spin trapping EPR provides a valuable tool for analyzing the production of OH•in vivo with high resolution in small tissue samples.