Fifty-two study participants underwent cervical spine surgery using intraoperative Iso-C imaging with or without spinal navigation. To evaluate prospectively the feasibility, advantages, limitations, and applications of Iso-C in cervical spine surgery. Existing stereotactic spinal navigational systems images must be acquired before surgery and typically require cumbersome point-to-point registration. Intraoperative computed tomography (CT) and magnetic resonance imaging (MRI) provide real-time information but can restrict access to the patient, preclude the use of traditional operating room tables, and are time-consuming. The Iso-C allows quick, CT-quality, real-time data acquisition without restricting access to the patient. The data acquired can be automatically transferred to navigational systems with the immediate ability to navigate for anterior or posterior cervical spine procedures. High-resolution isotropic three-dimensional data sets were acquired using the Iso-C intraoperative fluoroscopy in 52 cervical spine cases. In 30 cases, the data were imported automatically to the StealthStation Treon to support neuronavigation. In 22 cases, a postprocedural intraoperative CT was obtained with the Iso-C primarily to assess the extent of osseous decompression and/or the accuracy of implants or instrumentation. In most cases, a postoperative high-resolution CT image was obtained and compared with the Iso-C data. Successful automated registration suitable for navigation was attained for all anterior and posterior cervical spinal cases. The postprocedural intraoperative Iso-C data were 100% concordant with those of postoperative high-resolution CT as determined by a blinded neuroradiologist. Iso-C intraoperative fluoroscopy is an accurate and rapid way to perform CT-quality image-guided navigation in cervical spinal surgery. In most cases, it obviates the need for postoperative imaging.