Serum concentrations of cancer antigen (CA) 19-9 can be useful in monitoring response to therapy in pancreatic cancer. The objective of this study was to evaluate 5 automated CA 19-9 assays: ARCHITECT 12000 (Abbott Diagnostics, Abbott Park, IL), ADVIA Centaur (Bayer Diagnostics, Tarrytown, NY), UniCel Dxl 800 (Beckman Coulter, Fullerton, CA), IMMULITE 2000 (Diagnostic Products, Los Angeles, CA), and Elecsys E170 (Roche Diagnostics, Indianapolis, IN). All methods were evaluated for limit of detection, linearity, imprecision, method comparison, and reference intervals. Limit of detection results were all below 2.0 kU/L and met the manufacturers' claims. Linearity had deviation from target values that ranged from 4.5% to 26.7%. All methods showed acceptable imprecision with total coefficients of variation less than 8%. Method comparison by Passing-Bablok analysis resulted in slopes ranging from 1.00 to 2.06 and correlation coefficients of 0.85 to 0.98. Between 97.6% and 99.2% of results from healthy volunteers were less than 35 kU/L. All methods show acceptable analytic performance.