Despite recent instrumentation advancement in morphologic imaging, venous sampling remains a sensitive physiological investigation for functional localization of hormonal hypersecretion. The test requires technical expertise and reliable laboratory support, and should be selectively applied to address individual clinical problems in coordination with other imaging modalities. Depending on the venous anatomy, sampling may achieve regional localization or lateralization of the endocrine tumour. Adrenal venous sampling serves to lateralize functioning tumour, and to differentiate it from hyperplasia. Inferior petrosal sinus sampling confirms pituitary Cushing's disease and lateralizes the adenoma. Systemic venous sampling identifies the site of ectopic hormonal secretion. In the localization of pancreatic islet cell tumours, the test is further enhanced by sampling after intraarterial injection of hormonal secretagogue. The experience of venous sampling in a regional referral hospital is discussed, including the patient selection, its technical details, pitfalls and effectiveness in achieving tumour localization for patient management.