Background: Freehand single photon emission computed tomography (fSPECT) is a three-dimensional (3-D) tomographic imaging modality based on data acquisition with a handheld detector that is moved freely, in contrast to conventional, gantry-mounted gamma camera systems. In this pilot study, we evaluated the feasibility of fSPECT for intraoperative 3-D mapping in patients with parathyroid adenomas. Methods: Three patients (range 30 to 45 years) diagnosed with hyperparathyroidism (one primary and two tertiary) underwent parathyroid scintigraphy with technetium-99m sestamibi (99mTc-MIBI) to localize parathyroid adenomas. Two patients were referred with persistent hyperparathyroidism after conventional parathyroidectomy. In all three patients, a planar scintigraphy of the neck was performed 10 min after injection (p.i.) followed by SPECT/CT (Symbia T2, Siemens Healthcare) and a correlative ultrasound 2 h p.i. 99mTc-MIBI scan was performed the day before surgery in two patients and at the same day in one patient. fSPECT images were acquired intraoperatively using declipse SPECT (SurgicEyeTM). Results: A total of five parathyroid adenomas were successfully located with SPECT/CT. fSPECT allowed intraoperative detection of all adenomas, and successful parathyroidectomy was accomplished. Parathyroid hormone level decreased intraoperatively in all three patients, on average, by 79% (range 72% to 91%). Conclusion: In this preliminary study, we could demonstrate that intraoperative localization of parathyroid adenomas is feasible using the freehand SPECT technology, thus allowing an image-guided parathyroidectomy.