This study evaluated the accuracy of three-dimensional (3-D) ultrasound measurement of lymph node volume, and investigated the normal variations of cervical nodal volume. The volumes of 12 porcine neck lymph nodes were measured with 3-D ultrasound and calculated from two-dimensional (2-D) ultrasound using the ellipsoid formula, and compared with the volume measured by the water displacement method. 3-D ultrasound of cervical nodes was performed in 100 subjects, and the volumes of all ultrasound-detectable cervical nodes were measured. Results showed that the mean absolute errors of 3-D ultrasound and the 2-D ellipsoid formula method were 0.042 ml and 0.372 ml, respectively, and the mean percentage errors in measurement were 4.4% and 17.8%, respectively. In total, 830 nodes were detected in the subjects. Upper cervical nodes (0.48+/-0.47 ml) were significantly larger than submandibular nodes (0.39+/-0.24 ml) (p<0.05), and these two groups of lymph nodes were significantly larger than parotid (0.1+/-0.06 ml) and posterior triangle (0.1+/-0.11 ml) nodes (p<0.05). There was no significant variation of normal cervical node volume with advancing age (p>0.05). Men aged 20-29 years had significantly larger cervical nodes than women of the same age group (p<0.05), whereas there was no significant gender difference in nodal volume in other age groups (p>0.05). 3-D ultrasound measurement of cervical nodal volume is feasible. In vitro study showed that 3-D ultrasound had a lower mean error than the 2-D ellipsoid formula method in the measurement of cervical nodal volume. This study provides baseline information on the normal variations of cervical lymph node volume, as measured by 3-D ultrasound.