Abstract The role of preoperative and intraoperative procedures for the localization of insulinoma has been extensively debated. Transhepatic portal vein sampling before surgery has been recommended when other tests fail to localize the tumour. To determine the role of different investigations, 53 patients with insulinoma, four with hyperplasia or nesidioblastosis and one with insulin autoimmune syndrome were studied. Patients were operated on in three consecutive periods during each of which a different localization procedure was considered to represent the ‘gold standard’. During the first period, of 16 patients (including one with hyperplasia) investigated by arteriography, 13 underwent successful resection. Tumours in the other three patients with insulinoma were resected at a second operation, one during the first period and one each during the second and third periods. During the second period, 28 patients underwent exploration after transhepatic portal sampling: the tumour was found in all 26 patients with insulinoma operated on in this hospital, one patient with hyperplasia is receiving medical treatment and one patient had unsuccessful surgical exploration elsewhere despite positive findings on arteriography and transhepatic portal sampling performed in this department. During the third period 13 procedures were performed. All were successful using intraoperative ultrasonography without transhepatic portal sampling. In three further patients intraoperative localization failed because of non-adenomatous β cell disease. Left-sided resection successfully cured symptoms in two patients with hyperplasia and prompted the diagnosis of insulin autoimmune syndrome. High success rates for surgical treatment of insulinoma can be achieved with transhepatic portal vein sampling or intraoperative ultrasonography. Transhepatic portal sampling is therefore unnecessary before a first operation on the pancreas for insulinoma. In the rare failures of intraoperative localization of an insulinoma, a small left pancreatic resection can help to distinguish insulinoma from hyperplasia without precluding further segmental resection.