This experimental study was designed to compare two different fluoroscopy-based stereotactic surgical techniques for transcutaneous cervical pedicle screw (CPS) placement in the subaxial human cervical spine: (1) a custom-made aiming frame (AF) in combination with conventional fluoroscopy versus (2) a targeting device in combination with a computer-assisted image guidance system [i.e. virtual fluoroscopy (VF)]. Surgery was carried out on six preserved human total body specimens in a laboratory setting. Sixty pedicles (levels C3-C7) were measured in a multislice computed tomography (CT) image data set prior to surgery. Two groups consisting of three specimens and 30 pedicles each were defined according to the surgical technique. The AF consisted of radiolucent components with a fully adjustable arm for carrying the instruments necessary for placing the screws. The arm was angled according to the cervical pedicle axis, as determined by the preoperative CT scans and intraoperative lateral fluoroscopy. For VF, a targeting device was combined with a computer-assisted image-guided surgery unit. For both stereotactic techniques, 3.5 mm screws made of carbon fibre polyetheretherketone (ECF-PEEK) were inserted transcutaneously through stab incisions. Screw placement was assessed using a four-point grading system ranging from ideal (I) to unacceptable (III) where I = screw centred in pedicle, IIa = perforation of pedicle wall is less than one-fourth of the screw diameter, IIb = perforation of the pedicle wall is more than one-fourth of the screw diameter without contact to neurovascular structures, and III = CPS in contact with neurovascular structures. Fifty-eight pedicle screws could be evaluated without interfering metal artefacts according to the same CT protocol that was used preoperatively. The AF technique achieved a significantly smaller number of screws in contact with neurovascular structures compared with the VF technique (P = 0.021; Fisher's exact test) (Grade I n = 15; 64.3% AF vs. n = 13; 43.3% VF and Grade III n = 2; 7.1% AF vs. n = 10; 33.3% VF). Although neither of the two techniques was capable of completely preventing CPS perforations, transcutaneous CPS placement with a conventional fluoroscopy-based stereotactic AF can be considered a less expensive alternative to VF. This AF technique is able to reduce the number and severity of lateral pedicle wall violations compared to screw placement via the wide standard posterior open midline approach to the subaxial cervical spine. The results of this study are discussed in context with those obtained from different published modifications, since the first technical description of this surgical technique in 1994 by Abumi and co-workers.