OBJECTIVE The AlignRT3C system is an image-guided stereotactic positioning system (IGSPS) that provides real-time target localization. This study involves the first use of this system with three camera pods. The authors have evaluated its localization accuracy and tracking ability using a cone-beam computed tomography (CBCT) system and an optical tracking system in a clinical setting. METHODS A modified Rando head-and-neck phantom and five patients receiving intracranial stereotactic radiotherapy (SRT) were used to evaluate the calibration, registration, and position-tracking accuracies of the AlignRT3C system and to study surface reconstruction uncertainties, including the effects due to interfractional and intrafractional motion, skin tone, room light level, camera temperature, and image registration region of interest selection. System accuracy was validated through comparison with the Elekta kV CBCT system (XVI) and the Varian frameless SonArray (FSA) optical tracking system. Surface-image data sets were acquired with the AlignRT3C daily for the evaluation of pretreatment and interfractional and intrafractional motion for each patient. Results for two different reference image sets, planning CT surface contours (CTS) and previously recorded AlignRT3C optical surface images (ARTS), are reported. RESULTS The system origin displacements for the AlignRT3C and XVI systems agreed to within 1.3 mm and 0.7 degrees. Similar results were seen for AlignRT3C vs FSA. For the phantom displacements having couch angles of 0 degrees, those that utilized ART_S references resulted in a mean difference of 0.9 mm/0.4 degrees with respect to XVI and 0.3 mm/0.2 degrees with respect to FSA. For phantom displacements of more than +/- 10 mm and +/- 3 degrees, the maximum discrepancies between AlignRT and the XVI and FSA systems were 3.0 and 0.4 mm, respectively. For couch angles up to +/- 90 degrees, the mean (max.) difference between the AlignRT3C and FSA was 1.2 (2.3) mm/0.7 degrees (1.2 degrees). For all tests, the mean registration errors obtained using the CT_S references were approximately 1.3 mm/1.0 degrees larger than those obtained using the ART_S references. For the patient study, the mean differences in the pretreatment displacements were 0.3 mm/0.2 degrees between the AlignRT3C and XVI systems and 1.3 mm/1 degrees between the FSA and XVI systems. For noncoplanar treatments, interfractional motion displacements obtained using the ART_S and CT_S references resulted in 90th percentile differences within 2.1 mm/0.8 degrees and 3.3 mm/0.3 degrees, respectively, compared to the FSA system. Intrafractional displacements that were tracked for a maximum of 14 min were within 1 mm/1 degrees of those obtained with the FSA system. Uncertainties introduced by the bite-tray were as high as 3 mm/2 degrees for one patient. The combination of gantry, aSi detector panel, and x-ray tube blockage effects during the CBCT acquisition resulted in a registration error of approximately 3 mm. No skin-tone or surface deformation effects were seen with the limited patient sample. CONCLUSIONS AlignRT3C can be used as a nonionizing IGSPS with accuracy comparable to current image/marker-based systems. IGSPS and CBCT can be combined for high-precision positioning without the need for patient-attached localization devices.