Objective: A meta-analysis has not been previously performed to evaluate critically the diagnostic accuracy of endoscopic ultrasound-guided fine needle aspiration (EUS-FNA) of solely pancreatic ductal adenocarcinoma and address factors that have an impact on variability of accuracy. The aim of this study was to determine whether the presence of a cytopathologist, variability of the reference standard and other sources of heterogeneity significantly impacts diagnostic accuracy. Methods: We conducted a comprehensive search to identify studies, in which the pooled sensitivity, specificity, likelihood ratios for a positive or negative test (LR+, LR-) and summary receiver-operating curves (SROC) could be determined for EUS-FNA of the pancreas for ductal adenocarcinoma using clinical follow-up, and/or surgical biopsy or excision as the reference standard. Results: We included 34 distinct studies (3644 patients) in which EUS-FNA for a solid pancreatic mass was evaluated. The pooled sensitivity and specificity for EUS-FNA for pancreatic ductal adenocarcinoma was 88.6% [95% confidence interval (CI): 87.2-89.9] and 99.3% (95% CI: 98.7-99.7), respectively. The LR+ and LR- were 33.46 (95% CI: 20.76-53.91) and 0.11 (95% CI: 0.08-0.16), respectively. The meta-regression model showed rapid on-site evaluation (ROSE) (P = 0.001) remained a significant determinant of EUS-FNA accuracy after correcting for study population number and reference standard. Conclusion: EUS-FNA is an effective modality for diagnosing pancreatic ductal adencarcinoma in solid pancreatic lesions, with an increased diagnostic accuracy when using on-site cytopathology evaluation. © 2013 John Wiley & Sons Ltd.