Molecular mechanisms contributing to the tumorigenesis of pancreatic endocrine tumors (PETs) are still not well understood. Allelic deletions at chromosome 22q12.3 were detected in about 30-60% of PETs, suggesting that inactivation of one or more tumor suppressor genes on this chromosomal arm is important for their pathogenesis. Because the putative tumor suppressor gene tissue inhibitor of metalloproteinase-3 (TIMP-3) has been located at 22q12.3, we undertook a genetic analysis of TIMP-3 to determine its role in the tumorigenesis of PETs. Single-strand conformational polymorphism analysis, methylation-specific PCR, RNA expression analysis, and immunohistochemistry of TIMP-3 were performed in 21 sporadic PETs. Thirteen of 21 PETs (62%) revealed TIMP-3 alterations, including promoter hypermethylation and homozygous deletion. The predominant TIMP-3 alteration was promoter hypermethylation, identified in 8 of 18 (44%) PETs. It was tumor-specific and corresponded to loss or strong reduction of TIMP-3 protein expression. Notably, 11 of 14 (79%) PETs with metastases had TIMP-3 alterations, compared with only 1 of 7 (14%) PETs without metastases (P < 0.02). These data suggest a possibly important role of TIMP-3 in the tumorigenesis of human PETs, especially in the development of metastases, which has to be further evaluated in large-scale studies.