An experimental method is presented for determining the regioselectivity of deprotonation of unsymmetrical ketones in the gas phase. Mixtures of tautomeric enolate ions were prepared in a flowing afterglow apparatus and then assayed through a reaction with n-butyl nitrite in the collision cell of a triple quadrupole mass analyzer. Enolate ions were also prepared regioselectively by desilylation of the corresponding trimethylsilyl enol ethers with fluoride ion. Rate coefficients for the methanol-catalyzed tautomerization of the regioisomers were measured and were used to derive the equilibrium ratio of the tautomers. For 2-butanone it was found that the equilibrium mixture of enolate ions consisted of 55% of the more substituted isomer. For 3-methyl-2-butanone and 2-methyl-3-pentanone the equilibrium mixture comprised greater than 95% of the less substituted isomer. Several different bases were used to prepare nonequilibrium mixtures of enolate ions. Strong bases deprotonate these ketones irreversibly and in a statistical fashion. Deprotonation with hindered bases altered the composition of regioisomers only slightly. Ab initio molecular orbital calculations were performed on 2-butanone, 3-methyl-2-butanone, and their corresponding enolate ions at the MP4SDQ/ 6-31+G(d)//HF/6-31+G(d) level of theory. For 2-butanone, the calculations predict that the Z secondary enolate and the primary enolate have equal stabilities (Delta E < 0.1 kcal/mol), while the E secondary enolate is 4.1 kcal/mol higher in energy than the Z enolate ion. For 3-methyl-2-butanone, the tertiary enolate ion is calculated to be 4.3 kcal/mol higher in energy than the primary enolate ion. The computed gas-phase acidities of the two ketones are in excellent agreement with the experimentally determined values.