Carbon-bound hydrogen in sedimentary organic matter can undergo exchange over geologic timescales, altering its isotopic composition. Studies investigating the natural abundance distribution of ^1H and ^2H in such molecules must account for this exchange, which in turn requires quantitative knowledge regarding the endpoint of exchange, i.e., the equilibrium isotopic fractionation factor (α_(eq)). To date, relevant data have been lacking for molecules larger than methane. Here we describe an experimental method to measure α_(eq) for C-bound H positions adjacent to carbonyl group (H_α) in ketones. H at these positions equilibrates on a timescale of days as a result of keto-enol tautomerism, allowing equilibrium ^2H/^1H distributions to be indirectly measured. Molecular vibrations for the same ketone molecules are then computed using Density Functional Theory at the B3LYP/6-311G** level and used to calculate α_(eq) values for H_α. Comparison of experimental and computational results for six different straight and branched ketones yields a temperature-dependent linear calibration curve with slope = 1.081−0.00376T and intercept = 8.404−0.387T, where T is temperature in degrees Celsius. Since the dominant systematic error in the calculation (omission of anharmonicity) is of the same size for ketones and C-bound H in most other linear compounds, we propose that this calibration can be applied to analogous calculations for a wide variety of organic molecules with linear carbon skeletons for temperatures below 100 °C. In a companion paper (Wang et al., 2009) we use this new calibration dataset to calculate the temperature-dependent equilibrium isotopic fractionation factors for a range of linear hydrocarbons, alcohols, ethers, ketones, esters and acids.