Background/Aims: The secretin stimulation test is the principal diagnostic tool to identify Zollinger-Ellison syndrome (ZES). We investigated, by intra-individual comparison, which dose of secretin results in the highest diagnostic efficacy to identify the ZES. Methods: Fifty-seven paired secretin stimulation tests, using both 0.26 mug/kg and 0.78 mug/kg secretin, performed in 13 ZES patients and 12 controls, were analyzed and the findings confirmed in a validation cohort. Results: A gastrin increase of >100 ng/l was found to be the most sensitive and specific criterion for a positive test. Higher gastrin increases after 0.78 mug/kg compared to 0.26 mug/kg secretin contributed to a slightly more sensitive (82.9 vs. 80.5%) but less specific (68.8 vs. 81.3%) test. A validation cohort, with 98 tests using 0.26 mug/kg secretin in 21 ZES patients and 39 controls, provided similar results. In ZES patients with normal fasting serum gastrin levels (<100 ng/l), there was no diagnostic benefit from the use of a higher secretin dose. Conclusions: The 0.26 mug/kg secretin stimulation test has the best diagnostic efficacy for the ZES. and IAP.