Controversy continues to exist concerning the optimal diagnostic approach to a pancreatic head lesion suspected of being a neoplasm. The objective of this study was to evaluate the impact of needle biopsy in suspicious pancreatic head neoplasia and its effect on therapy and outcome. Seventy-three patients with symptoms or signs of periampullary neoplasia and a pancreatic head lesion identified on CT scan were reviewed retrospectively. Forty patients with potentially resectable lesions underwent intraoperative transduodenal core needle biopsy of the head of the pancreas. Thirty-three patients underwent CT-guided percutaneous fine-needle aspiration. The sensitivity and specificity of core needle biopsy were 76% and 100%, respectively. One death was directly related to the procedure and therapy was adversely affected in one patient with a false negative result. The sensitivity and specificity of percutaneous fine-needle aspiration were 85% and 92%, respectively, and were not significantly different from the core needle biopsy results (P >0.3). Three false negative fine-needle aspiration biopsies occurred in patients with potentially resectable lesions and a low clinical suspicion for malignancy. In patients with a mass in the head of the pancreas on CT scan, fine-needle aspiration biopsy offers results similar to those of intraoperative transduodenal core needle biopsy. In patients estimated to have resectable disease, a pancreaticoduodenectomy should be performed without a biopsy. For patients with unresectable disease, cytologic examination of fine-needle aspirate should be performed. If this examination is positive, it offers the advantage of facilitating the construction of a rational plan for palliation.