Recurrent biliary pain after cholecystectomy is presumably due to sphincter of Oddi dysfunction (SOD). There is no ideal non-invasive test for SOD, and the diagnosis often relies on invasive procedures such as sphincter of Oddi (SO) manometry. Amyl nitrite-augmented quantitative hepatobiliary scintigraphy (QHBS) was performed on nine asymptomatic volunteers and 22 patients with SOD of biliary types I and II. Normal QHBS parameters were established in the asymptomatic volunteers. QHBS revealed a partial obstructive pattern in nine patients in whom SO stenosis was suspected and in 13 patients in whom SO dyskinesia was suspected. This obstructive pattern remained unchanged in the former group, but was completely relieved in the latter group of patients on amyl nitrite administration. In conclusion, amyl nitrite-augmented QHBS proved to be a useful non-invasive method in the diagnosis of SOD of biliary types I and II and permitted differentiation between organic stenosis and functional motor abnormalities of the SO.