Biochar is emerging as one of the most promising tools to remove CO2 from the atmosphere and permanently sequester carbon (C) in soils. The technology is based on pyrolysis of fresh biomass, a process that also generates energy in the form of syn-gas and bio-oil. Pyrolysis conditions greatly affect both, biochar quality and energy yield. With regards to C sequestration, the ideal biochar product should be of highest possible recalcitrance in order to ensure its longevity in the soil. Due to the slow miner alization of biochar in soil environments, the assessment of its long-term stability and hence, C sequestration potential over long time periods (hundreds of years and more), presents a major scientific challenge. The overall objective of the present prop osal is to investigate the relationship between pyrolysis conditions, type of feedstock and biochar stability, and to combine advanced techniques for evaluation proxies of the long-term stability of biochar in soils. Transformations of biochar structures will be monitored in high precision incubations using 13C stable isotope labeling. The evolution of biochar chemical structures in soils will be monitored through black-carbon specific benzenepolycarboxylic acid biomarkers (BPCA) and 13C nuclear magnetic resonance (NMR). These time-consuming BPCA- and NMR- methods will be used to calibrate a high throughput mid-infrared spectroscopy method (MIR) for frequent monitoring of a large number of incubated samples. The physical structure of biochar will be moni tored with scanning electron microscopy. These advanced proxies will be combined with ecotoxicological tests and energy yield investigations to determine optimum pyrolysis conditions for mitigating greenhouse gas emissions with environmentally-safe biocha r.