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Locating the true flexion-extension axis of the knee can play an important role in component placement in a total knee arthroplasty, especially using contemporary computer-assisted surgical navigation. We determined if the commonly used transepicondylar axis is an accurate and reproducible substitute for the flexion-extension axis. Twenty-three fresh-frozen cadaveric distal femurs with intact soft tissue were imaged with computed tomography and reconstructed in three-dimensional virtual space. The transepicondylar axis was compared with a line equidistant from the articular surface of each femoral condyle. Measures were performed by three observers three times for each specimen. Interobserver and intraobserver variations were small, but the differences between axes were approximately 5 degrees. The difference between axes decreased when projected from three-dimensional space to traditional two-dimensional planes (coronal and transverse), explaining why this discrepancy has not been previously documented. The greater difference in three-dimensional space may account for midrange instability reported in total knee arthroplasty. The increased accuracy afforded by computer-assisted surgical navigation in total knee arthroplasty may be lost and increased malposition of components may occur if this discrepancy between reference axes is not appreciated and addressed.