Samples of groundwater, spring water and stream water contaminated by acid mine drainage (AMD), and uncontaminated stream water were collected and allowed to evolve in contact with air in the laboratory for 15–88 days. The objective of this study was to (1) document temporal changes in dissolved inorganic C (DIC) concentrations and stable isotopic composition (δ 13 C DIC ) and (2) to determine the reaction mechanism and resulting isotopic fractionation ( 13 C/ 12 C) accompanying the chemical evolution of AMD. The contaminated spring and stream samples and one groundwater sample (with no HCO3-) showed temporal decreases in pH, Fe 2+ , alkalinity, and DIC, and enrichment in δ 13 C DIC . One contaminated groundwater sample (with HCO3- between 529 and 630mg/L) showed a temporal increase in pH despite observed decreases in Fe 2+ , alkalinity and DIC, and enrichment in δ 13 C DIC . The uncontaminated stream samples showed a continuous temporal increase in pH, relatively constant alkalinity and DIC, and enrichment in δ 13 C DIC . The results suggest that proton production related to Fe 2+ transformation is the driving force for DIC loss in AMD-contaminated samples, and that DIC loss can be described by first order kinetics. The C isotope enrichment rates associated with DIC loss in the contaminated samples varied between 1.0‰ and 1.8‰ for stream water, 2.1‰ and 2.6‰ for the spring, 1.0‰ and 1.2‰ for groundwater with no HCO3-, and 7.6‰ and 9.3‰ for groundwater with high HCO3-. Variations in 13 C enrichment in the contaminated samples are attributed to differences in the initial Fe2+:HCO3- ratio. The effect of proton production on 13 C enrichment in the AMD-contaminated samples was modeled as a Rayleigh-type distillation, whereby isotope fractionation was constant and occurred in an “equilibrium closed system”. In the uncontaminated stream samples, C exchange between DIC and atmospheric CO 2 resulted in an overall enrichment in δ 13 C DIC of ∼6‰. It is concluded that C isotope enrichment induced by the chemical evolution of AMD in contaminated streams should range from 1.0‰ to 3.0‰ in the absence of in-stream processes that may affect DIC.