A novel tumor suppressor gene, PTEN/MMAC1, located on chromosome band 10q23.3, encodes a 403-amino acid, dual-specificity protein phosphatase. The defects in this gene are responsible for the development of some advanced cancers. Inactivating alterations, including mutations and deletions, in the PTEN/MMAC1 gene have been identified in several types of human cancers and cancer cell lines. To clarify the participation of the PTEN/MMAC1 gene in advanced gastric carcinogenesis, we screened their frequency of mutations in primary advanced gastric adenocarcinoma tissues. Cancer specimens and their corresponding normal tissues were obtained surgically from 60 patients with pathologically proven advanced gastric carcinoma at the Department of Surgery of Kaohsiung Medical University Hospital. All nine exons of the PTEN/MMAC1 gene were amplified using polymerase chain reaction and screened for mutations by single-strand conformation polymorphism analysis and followed by direct sequencing. After neutral polyacrylamide gel electrophoresis, 17 patients (28.3%) showed an apparent electrophoretic mobility shift between the cancer and its paired normal tissue. These results from direct sequencing indicated that mutations consisted of eight cases (47.1%) of missense mutation, five silent mutations (29.4%), two nonsense mutations (11.8%), a 12-bp deletion (5.9%), and a mutation within the splice donor site of intron 6 (5.9%). The mutation hot spots at codons 45, 66, 82 and 204 in advanced gastric cancer have not been observed previously. Based on the present analysis, our study implicated that the mutations of the PTEN/MMAC1 gene do not occur at a significant rate in human advanced gastric carcinoma, but the rare clustered mutation site (exons 2-6) perhaps suggested that PTEN/MMAC1 might contribute to the gastric carcinogenesis and its progression.