OBJECTIVE The authors undertook this cadaveric and angiographic study to examine the microsurgical anatomy of the V₃ segment of the vertebral artery (VA) and its relationship to osseous landmarks. A detailed knowledge of these variations is important when performing common neurosurgical procedures such as the suboccipital craniotomy and the far-lateral approach and when placing atlantoaxial instrumentation. METHODS A total of 30 adult cadaveric specimens (59 sides) were studied using magnification × 3 to × 40 after perfusion of the arteries and veins with colored silicone. Seventy-three vertebral angiograms were also analyzed. The morphological detail of the V₃ segment was described and measured in both the cadavers and angiograms. Transarticular screws were placed into 2 cadavers and the relationship of the trajectory to the V₃ segment was analyzed. RESULTS The authors identified 4 sites along the V₃ segment that are anatomically the most likely to be injured during surgical approaches to the craniovertebral junction. In 35% of the cadaveric specimens the vertical portion of V₃ formed a posteriorly oriented loop that could be injured during surgical exposures of the dorsal surface of C-2. The mean distance from the midline to the most posteromedial edge of the loop was 25.6 ± 3.5 mm (range 20-35 mm) on the left side and 30.4 ± 3.8 mm (range 23-36 mm) on the right side. On lateral angiograms, this loop projected posteriorly, with a mean distance of 9.8 ± 3.5 mm (range 0-15.7 mm) on the right side and 11.7 ± 1.2 mm (range 10-13.6 mm) on the left side. The horizontal segment of V₃ can be injured when exposing the lower lateral occipital bone and when the C-1 arch is exposed. The mean distance from the inferior border of the occipital bone to the superior surface of the horizontal segment of V₃ was 6 ± 2.8 mm on the right side and 5.6 ± 2.3 mm on the left. In 12% of cases the authors found no space between the horizontal portion of V₃ and the occipital bone. The medial edge of the horizontal segment of V₃ was located 23 ± 5.5 mm (range 10-30 mm) from the midline on the right side and 24 ± 5.7 mm (range 15-32 mm) on the left side. The transition between the V₂-V₃ segments after exiting the C-2 vertebral foramen is the most likely site of injury when placing C1-2 transarticular screws or C-2 pars screws. CONCLUSIONS The normal variation of the V₃ segment of the VA has been described with quantitative measurements. An awareness of the anatomical variations and the relationships to the surrounding bony anatomy will aid in reducing VA injury during suboccipital approaches, exposure of the dorsal surfaces of C-1 and C-2, and when placing atlantoaxial spinal instrumentation.