The aim of this study was to investigate the expression of hedgehog signaling molecules in gastric cancer. In situ hybridization, immunohistochemistry and RT-PCR for hedgehog signaling molecules, smoothened (SMO), suppressor of fused [Su(Fu)], and the target genes hedgehog-interacting protein (HIP) and platelet-derived growth factor receptor α (PDGFRα) were performed in 30 gastric cancer and two gastritis specimens. Using in situ hybridization, SMO expression was detected in 18/30 cancerous specimens (60%) as well as in 1/2 gastritis specimens (50%). Su(Fu) was expressed in 15/30 (50%), HIP in 14/30 (≈47%), and PDGFRα in 6/30 (20%) gastric cancer specimens. Despite the heterogeneous expression pattern, SMO, Su(Fu) and PDGFRα transcripts were highly correlated with the HIP transcript in the 30 gastric cancer specimens (p=0.0006, 0.0003 and 0.0441, respectively). Results from the in situ hybridization were further confirmed by RT-PCR for the expression of all of the genes and by immunohistochemistry for SMO expression. The findings revealed a set of genes for detecting Hh signaling activation in gastric cancer.