To understand the role of cyclooxygenase (COX)-2 in metastatic potential of oral cancer, COX-2 overexpressing KB/COX-2 cells were inoculated orthotopically into the masseter muscle or injected into the left cardiac ventricle of nude mice. KB/COX-2 showed about 4-fold increase of COX-2 protein expression as compared to KB/Neo which was a mock transfected control. In orthotopic inoculation, metastasis to the regional lymph nodes occurred in 2 out of 15 mice, and metastasis to the lung in 3 out of 15 mice. On the other hand, in intra-cardiac injection, hematogenous metastasis to the lung and bone occurred in 8 out of 10 mice in KB/COX-2, but no metastasis occurred except for only one metastasis to the femur bone out of 10 mice in KB/Neo. Treatment of KB/COX-2 with COX-2 small interfering RNA (siRNA) inhibited the colony formation but not cell growth in vitro, and suppressed tumorigenicity and hematogenous metastasis in nude mice. When expression of adhesion molecules such as E-cadherin, alpha-catenin, beta-catenin and CD44 was examined, there was no difference in alpha- and beta-catenin between the cells. However, expression of E-cadherin was detected in KB/Neo, but not in KB/COX-2. In contrast, expression of CD44 was markedly increased in KB/COX-2 as compared to KB/Neo. Treatment with COX-2 siRNA resulted in suppression of CD44 expression and detectable expression of E-cadherin in KB/COX-2. These findings suggested that overexpression of COX-2 increased hematogenous metastasis, at least in KB cells, via down-regulating E-cadherin and up-regulating CD44 expression.