Anatomic reference axes that determine rotational alignment of the tibial component have not been established. To assess variability of three anatomic reference axes (a new tibial anteroposterior axis that we proposed, the transmalleolar axis of the ankle, and the second metatarsus bone axis of the foot), we measured the angles between a defined anteroposterior axis of the tibia (a line perpendicular to the transepicondylar axis) and each of the three axes in 57 knees of healthy subjects using computed tomography scans. The angle between the defined anteroposterior axis and our proposed anteroposterior axis (a line connecting the middle of the posterior cruciate ligament and the medial edge of the patellar tendon attachment) averaged -0.2 degrees +/- 2.8 degrees (range, -5.5 degrees -6.3 degrees). The angle between the defined anteroposterior axis and the transmalleolar axis averaged 25.9 degrees +/- 9 degrees (range, 8 degrees -49.4 degrees), and the angle between the defined anteroposterior axis and the second metatarsus bone axis averaged 5.2 degrees +/- 10 degrees (range, -21.9 degrees -24 degrees). The variability of the anteroposterior axis was less than than the other reference axes. These data indicate that our proposed tibial anteroposterior axis is more reliable for determining rotational alignment of the tibial component in total knee arthroplasty.