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This study compared p53 expression with B7-H4, a novel cancer biomarker, in pancreatic ductal adenocarcinoma (PDA) resection specimens and in a pilot series of endoscopic ultrasound-guided fine-needle aspirations (EUS-FNAs).B7-H4 and p53 expression were evaluated by immunoperoxidase methods in 36 PDA and 15 EUS-FNA specimens and were scored for intensity and proportion of positive cells; cases were then assigned a final sum score.B7-H4 was detected in 33 (92%) of 36 PDA sections, 8 (89%) of 9 cytologically positive EUS-FNAs, and 1 (20%) of 5 cytologically negative EUS-FNAs. p53 was detected in 30 (83%) of 36 PDA sections, 4 (44%) of 9 cytologically positive EUS-FNAs, and 1 (20%) of 5 cytologically negative cases. One EUS-FNA case that was cytologically atypical but not diagnostic of malignancy expressed B7-H4 and p53. Some benign tissue components (intercalated cells/ducts, main pancreatic ducts, and acinar cells) were also positive for B7-H4 and/or p53. Overall expression of B7-H4 in benign tissues, however, was relatively low compared with that seen in most carcinoma cases.B7-H4 was expressed more often in PDA than was p53. Despite potentially problematic expression in benign/normal cells, the 2 markers target different cellular components and demonstrate potential diagnostic use for detection of PDA in resected and EUS-FNA specimens.