Neonatal hypoglycemia represents an emergency of heterogeneous etiology. The occurrence of persistent hypoglycemia caused by hyperinsulinism has not been well established. Some authors claim that it may be more common than previously suggested. The diagnostic goal is to distinguish hyperinsulinemia from other causes of hypoglycemia because management strategies differ. The diagnosis of persistent hypoglycemia attributable to hyperinsulinism is made when insulin secretion is excessive or inappropriate (> 10 microIU/ml). Medical management includes frequent feeding, high hydrocarbon intake, glucagon, diazoxide, somatostatin or steroid treatment. In case of resistance to medical intervention, surgery consisting of subtotal pancreatectomy is performed to avoid neurological sequelae. However, pediatric organic hypoglycemia secondary to hyperinsulinism can be caused by either diffuse or focal pancreatic lesions. Differentiation between these two types of lesion is necessary since partial pancreatectomy can prevent diabetes. In this prospective study, pancreatic venous sampling (PVS) was evaluated for the preoperative localization of lesions in 25 children with hyperinsulinism and correlated with surgical, pathological and clinical outcome. PVS is the most accurate preoperative technique for localizing focal lesions in children. Besides being safe and effective, it has the great advantage of detecting focal secretion, thus reducing the need for extensive surgery.