To propose a novel method for determining the three-dimensional (3D) root apex position of maxillary teeth using a two-dimensional (2D) panoramic radiograph image and a 3D virtual maxillary cast model. The subjects were 10 adult orthodontic patients treated with non-extraction. The multiple camera matrices were used to define transformative relationships between tooth images of the 2D panoramic radiographs and the 3D virtual maxillary cast models. After construction of the root apex-specific projective (RASP) models, overdetermined equations were used to calculate the 3D root apex position with a direct linear transformation algorithm and the known 2D co-ordinates of the root apex in the panoramic radiograph. For verification of the estimated 3D root apex position, the RASP and 3D-CT models were superimposed using a best-fit method. Then, the values of estimation error (EE; mean, standard deviation, minimum error and maximum error) between the two models were calculated. The intraclass correlation coefficient values exhibited good reliability for the landmark identification. The mean EE of all root apices of maxillary teeth was 1.88 mm. The EE values, in descending order, were as follows: canine, 2.30 mm; first premolar, 1.93 mm; second premolar, 1.91 mm; first molar, 1.83 mm; second molar, 1.82 mm; lateral incisor, 1.80 mm; and central incisor, 1.53 mm. Camera calibration technology allows reliable determination of the 3D root apex position of maxillary teeth without the need for 3D-CT scan or tooth templates.