Acidification of surface waters by acid mine drainage (AMD) contamination or atmospheric deposition perturbs the carbonate equilibrium, with unknown effects to the isotope ratio of dissolved inorganic carbon (DIC). We progressively acidified samples of NaHCO 3 , stream water, groundwater, and spring water contaminated by AMD (AMD spring) to a pH < 3 using H 2 SO 4 under open conditions (exposed to the atmosphere) and closed conditions (isolated from the atmosphere). The δ 13 C DIC of the acidified samples were enriched by 0.7‰ to 5.0‰ during the HCO 3 − dehydration phase and depleted by 0.6‰ to 2.3‰ during the phase after HCO 3 − was exhausted. The δ 13 CCO 2 showed enrichment and depletion trends that were similar to those of DIC. The positive enrichment in δ 13 C DIC during the HCO 3 − dehydration phase was driven by 1) kinetic fractionation of CO 2 during diffusion, or 2) a combination of fractionation accompanying HCO 3 − dehydration to CO 2(aq) followed by isotopic exchange of carbon between CO 2(aq) and HCO 3 − . We conclude that carbon isotope values in conjunction with concentrations of DIC species (CO 2(aq) , HCO 3 − , and CO 3 2− ) can be used to provide evidence for the effects of acidification on DIC in surface waters.