The concentration of glycosaminoglycan (GAG) in articular cartilage is known to be an important determinant of tissue mechanical properties based on numerous studies relating bulk GAG and mechanical properties. To date limited information exists regarding the relationship between GAG and mechanical properties on a spatially-localized basis in intact samples of native tissue. This relation can now be explored by using delayed gadolinium-enhanced MRI of cartilage (dGEMRIC—a recently available non-destructive magnetic resonance imaging method for measuring glycosaminoglycan concentration) combined with non-destructive mechanical indentation testing. In this study, three tibial plateaus from patients undergoing total knee arthroplasty were imaged by dGEMRIC. At 33–44 test locations for each tibial plateau, the load response to focal indentation was measured as an index of cartilage stiffness. Overall, a high correlation was found between the dGEMRIC index (T) and local stiffness (Pearson correlation coefficients r = 0.90, 0.64, 0.81; p < 0.0001) when the GAG at each test location was averaged over a depth of tissue comparable to that affected by the indentation. When GAG was averaged over larger depths, the correlations were generally lower. In addition, the correlations improved when the central and peripheral (submeniscal) areas of the tibial plateau were analyzed separately, suggesting that a factor other than GAG concentration is also contributing to indentation stiffness. The results demonstrate the importance of MRI in yielding spatial localization of GAG concentration in the evaluation of cartilage mechanical properties when heterogeneous samples are involved and suggest the possibility that the evaluation of mechanical properties may be improved further by adding other MRI parameters sensitive to the collagen component of cartilage. © 2004 Orthopaedic Research Society. Published by Elsevier Ltd. All rights reserved.