The relative stabilities of 2- and 3-acetylthiophenes have been evaluated by experimental thermochemistry and the results compared to high-level ab initio calculations. The enthalpies of combustion, vaporization, and sublimation were measured by rotating-bomb combustion calorimetry, Calvet microcalorimetry, correlation gas chromatography, and Knudsen effusion techniques and the gas-phase enthalpies of formation, at T = 298.15 K, were determined. Standard ab initio molecular orbital calculations at the G2 and G3 levels were performed, and a theoretical study on the molecular and electronic structures of the compounds studied has been conducted. Calculated enthalpies of formation using atomization and isodesmic reactions are compared with the experimental data. Experimental and theoretical results show that 2-acetylthiophene is thermodynamically more stable than the 3-isomer. A comparison of the substituent effect of the acetyl group in benzene and thiophene rings has been carried out.