Abstract Introduction Pre-clinical methods of delivering focal radiation to biological targets have made significant advances. Our group has developed the Small Animal Radiation Research Platform (SARRP), which is capable of using x-ray cone-beam CT (CBCT) to accurately guide delivery of conformal radiation. However, CBCT is deficient in localizing small orthotopic soft-tissue tumors. Bioluminescent imaging (BLI) is an affordable, accessible and sensitive ancillary method of detecting such tumors. In this study, we evaluate the setup accuracy of off-line BLI as a means to guide focal irradiation of small tumor volumes. Methods Athymic nude mice were used in accordance with institutional guidelines. Human pancreatic carcinoma cells (MiaPaCa-2) stably expressing luciferase were injected into a small glass bulb and sutured intra-abdominally to the tail of the pancreas in euthanized mice. Two methods of animal repositioning were employed during this process in which the animals were either unrestrained (n=6) or restrained (n=5). Two dimensional (2D) BLI and 3D bioluminescent tomography (BLT) were acquired of each supine mouse carcass. An ink-dot marked the “visual” centroid of the surface BL image. Carcasses were then transported to a SARRP for CBCT localization. Three methods of image guidance were applied. Using visual setup, the animal was positioned to align the ink-dot with the central axis of the SARRP. In the CBCT-aided setup, the pose of each carcass was adjusted to recapitulate its pose at the time of BLI. The 2D radial offset of the “true” bulb centroid in CBCT from the central axis of the SARRP was measured. In the 3D setup, the 3D contours of CBCT and BLT contours were registered and the 3D radial offset between the 2 centroids was calculated. Results Using the visual set up, single plane offsets of 2.2 mm +/- 1.2 mm and 2.5 mm +/- 1.5 mm were calculated between the isocenter of the vertical beam and the centroid of the bulb in unrestrained and restrained mice, respectively. After fusing the coronal 2D BL image to a corresponding coronal CBCT image, the central axis of the vertical beam was again aligned to the skin mark. The offset distances in a single plane were 2.2 mm +/- 1.2 mm and 2.4 mm +/- 1.1 mm in unrestrained and restrained mice, respectively. Finally, the offsets between the center of mass of the BL source and the CBCT glass bead after 3D fusion were 1.5 mm +/- 1 mm and 1.6 mm +/- 0.8 mm in unrestrained and restrained mice, respectively. Conclusion Restraining mice between modalities did not result in significant reduction of set up uncertainty. Similarly, use of 2D-BLI did not improve upon the visual set up method. The 3D-BLT fusion represents the best that can be achieved with off-line localization. An on-board approach with further improvement in BLT using prior information of anatomic and optical properties should be investigated to further improve localization uncertainty. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5725. doi:1538-7445.AM2012-5725