Preoperative parathyroid adenoma localization allows for a minimally invasive surgical approach. Current methods used for adenoma localization include ultrasound, sestamibi scan, and occasionally magnetic resonance imaging (MRI). In cases in which an adenoma is not localized after a radiologic work-up, the surgeon must perform a four-gland exploration. Preoperative selective venous sampling (SVS) has been described as a safe and clinically effective method to localize a parathyroid adenoma after failed exploration. The purpose of this study was to evaluate the efficacy of SVS as a primary localization technique in cases where conventional localization techniques have failed. Fourteen patients with nonlocalizing parathyroid adenomas after ultrasonography, sestamibi scanning, and MRI were retrospectively reviewed. Eight patients underwent SVS prior to surgery and six patients underwent a four-gland neck exploration without preoperative SVS. The two groups were assessed for accuracy of the SVS in localizing the adenoma, operative time, length of hospitalization, and complications. All of the patients in the study underwent successful uncomplicated surgical parathyroidectomy. The SVS proved accurate in preoperatively localizing the adenoma in 7 of 8 patients and allowed for a minimally invasive approach in 87% of patients. The mean operative time was 33 minutes for the SVS group and 67 minutes in the non-SVS group. None of the patients in either group experienced procedural complications. This study suggests that preoperative SVS may represent a safe and effective method of preoperatively localizing the nonlocalized parathyroid adenoma. In cases where conventional radiologic techniques fail to localize an adenoma, SVS may obviate the need for a four-gland exploration.