Metastasis is the chief cause of mortality in cancer patients. Recently, chemokines and chemokine receptors were shown to play an important role in the metastasis of various cancers. We examined the role of chemokine receptor-mediated signaling in the invasion potential of human oral squamous cell carcinoma (OSCC) cell lines that were derived from 5 primary tumors and 6 cervical lymph node metastases. Comprehensive analysis of the mRNAs for human chemokine receptors showed that the OSCC cell lines had uniform expression patterns of chemokine receptors. Overall, there were no consistent differences in the expression of chemokine receptors between primary site- and lymph node metastasis-derived cell lines. However, a highly invasive OSCC cell line (SAS-H1) expressed up-regulation of CCR5, CCR6, CCR7, CXCR1, CXCR6 and CX3CR1 compared to a poorly invasive OSCC cell line (SAS-L1). Then we examined whether factors in the tumor microenvironment regulated chemokine receptor expression in SAS-H1 cells. Specifically, transforming growth factor (TGF) -β1 enhanced the expression of CCR5, CCR6, CCR7 and CX3CR1. Pretreatment of SAS-H1 cells with transforming growth factor (TGF) -β1 increased the expression of CCR7 and CX3CR1, and then enhanced CCL21- and CX3CL1-induced directional migration (1.5-fold enhancement as compared with untreated control). In addition, CX3CL1 increased the adhesion of SAS-H1 cells on uncoated tissue culture plates. Neither chemokine stimulated cell proliferation. Treatment of SAS-H1 cells with CX3CL1 activated the phosphotidylinositol-3-kinase (PI3K) and MEK signal transduction pathways. Our results suggest that chemokine receptor-mediated signaling is involved in the local invasion and metastasis of human OSCC.