OBJECTIVE To assess the effect of timing of intraoperative parathormone (iPTH) samples and 25-hydroxyvitamin D (25-OHD) status on decision-making during parathyroidectomy. METHODS A total of 77 patients with primary hyperparathyroidism and iPTH levels (preincision, preremoval, 5 (T5) and 10 (T10) minutes postremoval) performed during parathyroidectomy were reviewed. RESULTS Forty-one percent of patients were 25-OHD insufficient. We noted a significant correlation between preoperative 25-OHD and preincision iPTH (P=0.002) but not iPTH at postremoval levels (T5, P=0.89; T10, P=0.42). When compared with preincision iPTH, the use of either the higher preincision or preremoval iPTH baseline significantly improves the assay sensitivity from 83% to 93% at T5 (P=0.01) and 87% to 97% at T10 (P=0.02). Surgical cure was obtained in 98% of patients. CONCLUSION Obtaining preremoval iPTH allowed earlier decision with respect to operative completion in 38% of cases. 25-OHD status does not appear to significantly affect interpretation of iPTH levels. SIGNIFICANCE Obtaining both baseline levels significantly improves sensitivity in iPTH monitoring.