Skeletal muscle volume is an important indicator of muscle function. Three-dimensional (3D) freehand ultrasound provides a noninvasive method for determining muscle volume and is acquired using a standard clinical ultrasound machine and an external tracking system to monitor transducer position. Eleven healthy volunteers were scanned with a 3D freehand system that uses an optical tracking device. Interest was concentrated on one of the muscles of the quadriceps group, rectus femoris and volume measurements performed on 30 mm cross-sections were compared with measurements derived from magnetic resonance imaging. Measured muscle volumes ranged from 5 cm(3) to 28 cm(3). The mean difference between measurements from 3D freehand ultrasound and magnetic resonance was 0.53 cm(3) with 95% limits of agreement of +/-2.14 cm(3). Muscle volume measurements obtained using 3D ultrasound were within +/-16% of the corresponding value from magnetic resonance imaging. We have shown for the first time that 3D freehand ultrasound can be used to determine human skeletal muscle volume accurately in vivo.