Background: The clinical management of pancreatic cancer is severely hampered by the absence of effective screening tools. Methods: Sixty-seven biomarkers were evaluated in prediagnostic sera obtained from cases of pancreatic cancer enrolled in the Prostate, Lung, Colorectal, and Ovarian Cancer Screening Trial (PLCO). Results: The panel of CA 19-9, OPN, and OPG, identified in a prior retrospective study, was not effective. CA 19-9, CEA, NSE, bHCG, CEACAM1 and PRL were significantly altered in sera obtained from cases greater than 1 year prior to diagnosis. Levels of CA 19-9, CA 125, CEA, PRL, and IL-8 were negatively associated with time to diagnosis. A training/validation study using alternate halves of the PLCO set failed to identify a biomarker panel with significantly improved performance over CA 19-9 alone. When the entire PLCO set was used for training at a specificity (SP) of 95%, a panel of CA 19-9, CEA, and Cyfra 21-1 provided significantly elevated sensitivity (SN) levels of 32.4% and 29.7% in samples collected <1 and >1 year prior to diagnosis, respectively, compared to SN levels of 25.7% and 17.2% for CA 19-9 alone. Conclusions: Most biomarkers identified in previously conducted case/control studies are ineffective in prediagnostic samples, however several biomarkers were identified as significantly altered up to 35 months prior to diagnosis. Two newly derived biomarker combinations offered advantage over CA 19-9 alone in terms of SN, particularly in samples collected >1 year prior to diagnosis. However, the efficacy of biomarker-based tools remains limited at present. Several biomarkers demonstrated significant velocity related to time to diagnosis, an observation which may offer considerable potential for enhancements in early detection. © 2014 Nolen et al.