Traditionally, a 2-step protocol has been used for deep biliary cannulation. The purpose of the present prospective study was to find out the feasibility and safety of the novel sequential 3-step protocol (traditional cannula with guidewire, double-guidewire, and needle-knife techniques) for deep biliary cannulation. All consecutive patients admitted for endoscopic retrograde cholangiopancreatography (ERCP) to a single, very experienced ERCP endoscopist during the year 2009 with intended biliary cannulation and with unhindered access to a native papilla (n=105) were included in the present study. The overall success rate for deep biliary cannulation was 99% (104/105). Cannulation with cannula and guidewire was attempted in all patients and proved successful in 80% (84/105) of the attempts, the double-guidewire technique was applied in 19% (20/105) and was successful in 65% (13/20) of the cases, and the needle-knife technique was applied in 7% (7/105) with success in all cases. The median cannulation time was 1 minute (range, 0 to 27 min). The rate of post-ERCP pancreatitis was 3% (3/105) and post-ERCP cholangitis 2% (2/105). We conclude that in experienced hands, the novel sequential 3-step protocol for biliary cannulation tested herein proved to be an effective cannulation protocol with the overall success rate of 99%. The complication rate of these ERCP procedures (5%) was within acceptable limits.