It is generally expected that the kinetics of reactions inside living cells differs from the situation in bulk solutions. Macromolecular crowding and specific binding interactions could change the diffusion properties and the availability of free molecules. Their impact on reaction kinetics in the relevant context of living cells is still elusive, mainly because the difficulty of capturing fast kinetics in vivo. This article shows spatially resolved measurements of DNA hybridization kinetics in single living cells. HeLa cells were transfected with a FRET-labeled dsDNA probe by lipofection. We characterized the hybridization reaction kinetics with a kinetic range of 10 micros to 1 s by a combination of laser-driven temperature oscillations and stroboscopic fluorescence imaging. The time constant of the hybridization depended on DNA concentration within individual cells and between cells. A quantitative analysis of the concentration dependence revealed several-fold accelerated kinetics as compared with free solution for a 16-bp probe and decelerated kinetics for a 12-bp probe. We did not find significant effects of crowding agents on the hybridization kinetics in vitro. Our results suggest that the reaction rates in vivo are specifically modulated by binding interactions for the two probes, possibly triggered by their different lengths. In general, the presented imaging modality of temperature oscillation optical lock-in microscopy allows to probe biomolecular interactions in different cell compartments in living cells for systems biology.