The electromyographic activity of eight muscles of the rotator cuff and shoulder girdle (supraspinatus, infraspinatus, subscapularis, pectoralis, latissimus dorsi, and the anterior, middle, and posterior deltoid) was measured from the nondominant shoulders of 11 subjects during a series of 29 isometric contractions. The contractions simulated different positions used for strength testing of the rotator cuff and involved elevation, external rotation, and internal rotation at three degrees of initial humeral rotation (-45 degrees of internal rotation, 0 degree, +45 degrees of external rotation) and scapular elevation (0 degree, 45 degrees, 90 degrees). Isolation of the supraspinatus muscle was best achieved with the test position of elevation at 90 degrees of scapular elevation and +45 degrees (external rotation) of humeral rotation. Isolation of the infraspinatus muscle was best achieved with external rotation at 0 degree of scapular elevation and -45 degrees (internal rotation) of humeral rotation. Isolation of the subscapularis muscle was best achieved with the Gerber push-off test. This study used four criteria for identifying the optimal manual muscle test for each rotator cuff muscle: 1) maximal activation of the cuff muscle, 2) minimal contribution from involved shoulder synergists, 3) minimal provocation of pain, and 4) good test-retest reliability. Based on the results of this study and known painful arcs of motion, an objective identification of the optimal tests for the manual muscle testing of the cuff was elucidated.