Imaging of the brachial plexus with MRI and standard two-dimensional (2D) ultrasound has been reported, and 2D ultrasound-guided regional anaesthetic block is an established technique. The aim of this study was to map the orientation of the brachial plexus in relation to the first rib, carotid and subclavian arteries, using three-dimensional (3D) ultrasound. A free-hand optically tracked 3D ultrasound system was used with a 12 MHz transducer. 10 healthy volunteers underwent 3D ultrasound of the neck. From the 3D ultrasound data sets, the outlines of the brachial plexus, subclavian artery and first rib were manually segmented. A surface was interpolated from the series of outlines to produce a spatially orientated 3D reconstruction of the brachial plexus. The brachial plexus could be mapped in all volunteers, although a variation in image resolution between individuals existed. Anatomical variations were demonstrated between the 10 volunteers; the most notable and clinically relevant was the alignment of the plexus divisions. 3D reconstructions illustrated the plexus, changing its orientation from a vertical alignment in the interscalene region to a more horizontal alignment in the supraclavicular fossa. Spatial mapping of the brachial plexus is possible with 3D ultrasound using the subclavian artery and first rib as landmarks. There is a deviation from the conventionally described anatomy and this may have implications for the administration of regional anaesthesia.