BACKGROUND Patellofemoral pain syndrome is one of the most common knee problems and may be related to abnormal patellar tracking. Our purpose was to compare, in vivo and noninvasively, the patellar tracking patterns in symptomatic patients with patellofemoral pain and those in healthy subjects during squatting. We tested the hypothesis that patients with patellofemoral pain exhibit characteristic patterns of patellar tracking that are different from those of healthy subjects. METHODS Three-dimensional patellar kinematics were recorded in vivo with use of a custom-molded patellar clamp and an optoelectronic motion capture system in ten healthy subjects and nine subjects with patellofemoral pain. The position of osseous knee landmarks was digitized while subjects stood upright, and then patellofemoral kinematics were recorded during squatting. The tracking technique was validated with use of both in vitro and in vivo methodologies, and the average absolute error was <1.2 degrees and <1.1 mm. RESULTS At 90 degrees of knee flexion, the patella showed lateral spin (the distal pole of the patella rotated laterally) in subjects with patellofemoral pain (mean and standard deviation, -10.13 degrees +/- 2.24 degrees) and medial spin in healthy subjects (mean, 4.71 degrees +/- 1.17 degrees) (p < 0.001). At 90 degrees of knee flexion, the patella demonstrated significantly more lateral translation in subjects with patellofemoral pain (mean, 5.05 +/- 3.73 mm) than in healthy subjects (mean, -4.93 +/- 3.93 mm) (p < 0.001). CONCLUSIONS Kinematic differences between healthy subjects and subjects with patellofemoral pain were demonstrated through a large, dynamic range of knee flexion angles. Increased lateral patellar translation and lateral patellar spin in subjects with patellofemoral pain suggest that the patella is not adequately balanced during functional activities in this group. Prospective studies are needed to identify when patellofemoral pain-related changes begin to occur and to determine the risk for the development of patellofemoral pain in individuals with abnormal kinematics.