Knowledge of anterior-posterior (A-P) tibial contact locations provides an objective assessment of the relative motion of the tibia on the femur following total knee arthroplasty (TKA), which can be used to compare the effects of different components, surgical techniques, and alignment goals on knee function in vivo. Both the lowest point method and the penetration method have been used to calculate A-P tibial contact locations using three-dimensional (3D) model to two-dimensional (2D) image registration. The primary objective of this study was to quantify errors in calculating the A-P tibial contact location using the lowest point and penetration methods because the errors in calculating the A-P tibial contact locations using these two methods are unknown. The A-P tibial contact locations were calculated with the two methods and simultaneously measured with a tibial force sensor in ten fresh-frozen cadaveric knee specimens with a TKA. Single-plane radiographs of the knee specimens were acquired at 0 deg, 30 deg, 60 deg, and 90 deg of flexion in neutrally, internally, and externally rotated orientations. While the radiographs were exposed, reference A-P tibial contact locations were simultaneously collected using the tibial force sensor to be compared to the calculated A-P tibial contact locations. The overall root-mean-squared-errors (RMSEs) in the A-P tibial contact location calculated with the lowest point method, the penetration method with penetration, and penetration method without penetration were 5.5 mm, 3.6 mm, and 8.9 mm, respectively. The overall RMSE was lowest for the penetration method with penetration, making it the superior method for calculating A-P tibial contact locations.