Accurate quantification of different soil organic carbon (SOC) fractions is needed to understand their relative importance in the global C cycle. Among the chemical methods of SOC fractionation, oxidative degradation is considered more promising because of its ability to mimic the natural microbial oxidative processes in soil. This study focuses on detailed understanding of changes in structural chemistry and isotopic signatures of SOC upon different oxidative treatments for assessing the ability of these chemicals to selectively isolate a refractory fraction of SOC. Replicated sampling (to ~1 m depth) of pedons classified as Typic Fragiudalf was conducted under four land uses (woodlot, grassland, no-till and conventional-till continuous corn [Zea mays L.]) at Wooster, OH. Soil samples (<2 mm) were treated with three oxidizing agents (hydrogen peroxide (H2O2), disodium peroxodisulfate (Na2S2O8) and sodium hypochlorite (NaOCl)). Oxidation resistant residues and the bulk soil from A1/Ap1 horizons of each land use were further analyzed by solid-state 13C nuclear magnetic resonance (NMR) spectroscopy and accelerator mass spectrometry to determine structural chemistry and 14C activity, respectively. Results indicated that, oxidation with NaOCl removed significantly less SOC compared to Na2S2O8 and H2O2. The NMR spectra revealed that NaOCl oxidation preferentially removed lignin-derived compounds at 56 ppm and at 110-160 ppm. On the other hand, the SOC resistant to Na2S2O8 and H2O2 oxidation were enriched with alkyl C groups, which dominate in recalcitrant macromolecules. This finding was corroborated by the 14C activity of residual material, which ranged from -542 to -259‰ for Na2S2O8 resistant SOC and -475 to -182‰ for H2O2 resistant SOC as compared to relatively greater 14C activity of NaOCl resistant residues (-47 to 61‰). Additionally, H2O2 treatment on soils after light fraction removal was more effective in isolating the oldest (14C activity of -725 to -469‰) SOC fraction. The Δ14C signature of SOC removed by different oxidizing agents, calculated by mass balance, was more or less similar irrespective of the difference in labile SOC removal efficiency. This suggests that SOC isolated by many fractionation methods is still a mixture of much younger and older material and therefore it is very important that the labile SOC should be completely removed before measuring the turnover time of stable and refractory pools of SOC. © 2009 Springer Science+Business Media B.V.