Recent studies suggest that hedgehog (HH)-pathway signaling is required for the initiation and continued growth of pancreatic adenocarcinoma (PAC). Definitive gene expression analysis of PAC remains difficult, owing to the host desmoplastic stromal interaction and subsequent tumor heterogeneity. The primary goal of this study was to evaluate the effect of heterogeneity within a series (n=5) of matched clinical PAC biopsies [snap-frozen, formalin-fixed paraffin-embedded (FPE), endoscopic ultrasound-guided fine-needle aspirate (EUS-FNA)]. Differential expressions, specific to tumor cells, were evaluated by comparisons of uninvolved pancreas (n=9), EUS-FNA (n=14), and macrodissected (tumor-cell-enriched) biopsies (n=16). To determine whether treatment modulates gene expression, a unique (independent) set of synchronous EUS-FNA samples (n=4) was obtained before, and 2 weeks after, chemoradiation. mRNA levels were evaluated using real-time quantitative polymerase chain reaction formatted in a TaqMan low-density array, which was capable of simultaneously quantifying 46 independent genes in the HH pathway. Protein levels for Patched, Smoothened, and glioma-associated oncogene 1 (Gli-1) in FPE tissues were determined, using immunohistochemistry. A significant concordance (P<0.0001) was observed in the HH-pathway mRNA levels between matched surgically resected (both snap-frozen and FPE) and EUS-FNA biopsies. HH-pathway mRNA levels changed (increased) only after macrodissection, suggesting localization to tumor cells. Immunohistochemical staining for Patched, Smoothened, and Gli-1 confirmed the increased (P<0.001) levels of protein in the PAC cells, compared with cells from uninvolved pancreas. EUS-FNA biopsies that were obtained before and during chemoradiation demonstrated no significant changes in HH-pathway gene expression. Collectively, these studies demonstrate presence of HH-pathway expression in all the clinical PAC biopsies examined, suggesting that this is a significant tumor-associated target and offering the possibility that specific molecular profiling might be attempted from these heterogeneous tissues.