Previous laboratory measurements showed topographical variation in the properties of articular cartilage in several animal species and in humans. In this study we characterize for the first time the topographical variation of the stiffness of the human knee articular cartilage in vivo using a novel arthroscopic indentation instrument. The instrument indicates the stiffness in the form of force (Newtons) by which the tissue resists the constant deformation (300 m) produced by the small (1-mm diameter) cylindrical indenter. Measurements were carried out at eight sites in the knee joint of 20 persons who had intact cartilage in the arthroscopic examination. The stiffest cartilage (5.6 ؎ 1.2 N, mean ؎ SD) was located in the lateral condyle of the femur, whereas the softest cartilage (2.4 ؎ 0.8 N) was in the medial plateau of the tibia. In general, the cartilage stiffness was higher in the femur than in the tibia (p < 0.01) or in the patella (p < 0.01). In the femur, the condyles were stiffer than the patellar surface (p < 0.01). The stiffness variation was consistent with earlier biphasic indentation analyses of laboratory measurements with the knee joint cartilage of cadavers. This study provides baseline data for characterization of cartilage stiffness in pathological situations or follow-up of the cartilage quality after surgical interventions.