Background: Frozen section analysis has traditionally been used to confirm the identity of parathyroid tissue intraoperatively; however, it is time-consuming and costly and requires the excision of a significant portion of tissue. An intraoperative biopsy and analysis with a parathyroid hormone (PTH) assay is a possible alternative; however, this technique has not been perfected. Methods: Two hundred twenty-three tissue specimens were collected prospectively from patients undergoing neck exploration. Each specimen was sampled intraoperatively using three different biopsy techniques: a fine-needle aspiration (FNA) with 10 passes of a needle (FNA10), a FNA with 20 passes of a needle (FNA20), and a tissue biopsy of approximately 1.0 mm3 (BIOPSY). The PTH concentration of each sample was determined via the Elecsys® 1010 PTH immunoassay. The final tissue diagnosis was determined by histology or operative data. Results: Parathyroid samples from all techniques had higher median PTH concentrations than nonparathyroid samples. However, the accuracies for the detection of parathyroid tissue varied markedly (PTH cutoff of 1000 pg/ml): the accuracies of the FNA10 and FNA20 were 71 and 80%, respectively, while the BIOPSY was 99% accurate. Conclusions: This is the first prospective study evaluating multiple methods to diagnose parathyroid tissue intraoperatively using a rapid PTH assay. We conclusively show that the BIOPSY technique is 99% accurate for the diagnosis of parathyroid tissue, and therefore, should be the method of choice when the intraoperative confirmation of parathyroid tissue is needed. © 2006 Elsevier Inc. All rights reserved.