Background: In the absence of precut needle-knife sphincterotomy (NKS), failure of biliary cannulation may occur in up to 10% of cases. There are few prospective evaluations of the safety and efficacy of NKS, and studies of its early use in difficult cannulation have been inconclusive. Whether precut NKS after failure of primary biliary cannulation is independently associated with post-ERCP pancreatitis (PEP) remains controversial. Objective: To examine the relationship between NKS and PEP. Design: Analysis of prospectively collected data from two randomized trials of ERCP techniques, with PEP as the primary endpoint measure. Setting: Tertiary-care academic medical center. Patients: This study involved 732 patients from two successive, prospective, randomized trials of naïve papilla cannulation between November 2001 and April 2006. Patients with pancreatic or ampullary cancer were excluded. Intervention: Naïve papilla cannulation, NKS, primary guidewire versus contrast-assisted cannulation, and glyceryl trinitrate patch versus placebo. Main Outcome Measurements: PEP and procedure-related complications. Results: NKS was performed in 94 of 732 patients (12.8%) and was successful in achieving bile duct access in 80 of 94 patients (85%). Cannulation success in the entire group was 717 of 732 patients (97.7%). The overall frequency of PEP following NKS was 14.9% (14 of 94 patients) compared with 6.1% (39 of 638 patients) without NKS (P < .001). The incidence of PEP increased with an increasing number of attempts at cannulating the papilla. Pancreatic stents were inserted in 22 patients, 5 of whom developed pancreatitis. In multivariate analysis, independent predictors of PEP were as follow: female sex (odds ratio [OR] = 3.5, P = .028), suspected sphincter of Oddi dysfunction (SOD) (OR = 9.7, P < .001), partial pancreatic drainage (OR = 4.8, P = .011), 10 to 14 attempts at papilla cannulation (OR = 4.4, P = .031), and ≥15 attempts at papilla cannulation (OR = 9.4, P = .013). NKS was not an independent predictor of PEP. There were no perforations, no major bleeding, and no cases of severe pancreatitis in the NKS group. Limitations: Nonrandomized for NKS intervention. Conclusions: The number of attempts at cannulating the papilla is independently associated with PEP, and the risk increases with an increasing number of attempts. NKS is not an independent predictor of PEP.