In radiotherapy planning, the calibration of CT number in terms of electron density (electrons per cm3) is essential for dose computations in heterogeneous tissue. For electron density of soft tissues with low atomic numbers (6 < Z < 8) and with kilovoltage (kV) CT number values below 100, the calibration curve is linear and well justified theoretically. For tissues with higher CT number values (> 100), empirical methods based on phantom experiments with bone‐substitute materials have been performed to produce an approximate bone calibration curve. This approach was first validated for in‐vivo tissues by plotting kV‐CT numbers against the electron density measured independently by Compton‐scatter imaging. With the advent of megavoltage (MV) CT scanning on tomotherapy machines, we now have a similar opportunity for the measurement of electron densities in vivo. In this work, we have scanned radiotherapy patients (N ∼ 30) using a kilovoltage CT scanner (Picker PQ5000, Marconi Medical Systems, Cleveland, Ohio) operated at 130 kVp and we repeated the studies using 3.5 MV X‐rays produced by a tomotherapy unit (TomoTherapy Inc.). Through manual identification of common anatomical features and sampling of homologous tissue regions, a calibration curve of kV‐CT number versus electron density was plotted. The curve, which includes 50 regional data points per patient, shows very good agreement with previously reported empirical equations. This study reaffirms that kV‐CT scanning, done at one fixed energy (e.g. 130 kVp), can be used for tissue electron densitometry in living soft tissues and bone. © 2005, American Association of Physicists in Medicine. All rights reserved.