The purposes of this study were to evaluate the reliability and validity of ultrasound (US) for measuring the cross-sectional area (CSA) of the longus colli (LC) as compared with magnetic resonance imaging (MRI), and to determine the change in CSA of the LC during contraction. 27 healthy volunteers participated in the study. In order to assess the validity of US, the US measurements of the CSA of the LC were compared to those determined with MRI. Two testers established the measurements to ascertain intra- and interrater reliability. The widely spaced limits of agreement (2SD=+/-0.45) reflect the large variability between the measurements by US and MRI. The ICC for the intra- and interrater reliability for the CSA of the LC was respectively 0.71 (95% CI, 0.57-0.81; SEM, 0.17; SDD, 0.48) and 0.68 (95% CI, 0.48-0.81; SEM, 0.18; SDD, 0.50). The CSA of the LC increased significantly during contraction of the LC (p=0.006). Results from this study show that the validity and reliability of US to evaluate the CSA of the LC is questionable, which may be due to both anatomical characteristics and methodological limitations.