High accuracy in absolute dose measurements is required in radiation therapy and dosimetric protocols require the use of air ionization chambers. These protocols involve chamber specific calibration factors, which depend fundamentally upon the chamber volume. In this work, we investigate the ability of micro‐computed tomography (micro‐CT) to determine differences in the chamber volume of four nominally identical Exradin A1SL cylindrical ion chambers. A GE Locus micro‐CT imaging system (General Electric Healthcare, London, Ontario) was used to acquire 20 micron resolution images of the four ion chambers. The ionization response of each chamber in a 10×10cm2 10 MV x‐ray reference field was also measured, and the readings were corrected for polarity and ion recombination effects. GE MicroView software (Version 2.1.1) and its semi‐automatic pixel volume measurement option was used to determine the sensitive air volume of the ion chambers. Both the ionization response of the chambers and the volumes were normalized to the chamber with the largest values. The relative differences in response and volume for each chamber agreed within 1%. Micro‐CT is a promising tool for the accurate determination of chamber volume, and potentially for the independent determination of chamber calibration factors. © 2006, American Association of Physicists in Medicine. All rights reserved.