The acromioclavicular joint (ACJ) is a common source of pain and disability. Several provocative tests are used to diagnose ACJ lesions. The aim of this study was to evaluate anatomic changes of the ACJ using dynamic ultrasonography during provocative tests. Eighty ACJs of 40 healthy volunteers were subjected to dynamic ultrasonographic examinations. Examinations were performed in a resting position and during passive external rotation, cross body adduction, and active compression testing. Deep joint space distance and capsule displacement were measured ultrasonographically. Mean deep joint space distances during cross body adduction (6.4 +/- 1.6 mm) and active compression (6.6 +/- 1.8 mm) testing were significantly smaller than during passive external rotation testing (7.7 +/- 1.7 mm, P < 0.01) or rest (8.1 +/- 1.8 mm, P < 0.01). Mean capsule displacements during active compression (3.5 +/- 0.8 mm) and cross body adduction (3.4 +/- 0.7 mm) testing were significantly greater than those during passive external rotation testing (2.9 +/- 0.7 mm, P < 0.01) or rest (2.5 +/- 0.7 mm, P < 0.01). Dynamic ultrasonography revealed that the cross body adduction and active compression tests applied more stress to the ACJ than the passive external rotation test, which suggests that these two tests are likely to be more useful provocative tests for the diagnosis of ACJ lesions. Furthermore, the dynamic properties of ultrasonography could be used to estimate the suitability and validity of provocative tests in other musculoskeletal diseases.