Metastasis is a complex, multistep process by which a cancer cell leaves the primary tumor, travels to a distant site via the circulatory system, and establishes a secondary cancer. A deeper understanding of the molecular events underlying metastasis will provide information that will be useful for the development of new diagnostic and therapeutic strategies. The B16 and B16F10 mouse melanoma cell lines are widely used as model system for studying many aspects of cancer biology including metastasis. Compared with B16, which has a low metastatic potential, the highly metastatic cell line B16F10 displayed a higher metastatic ability along with higher expression levels of the metastasis-associated phosphatase of regenerating liver-3 (PRL-3). B16 cells transfected with PRL-3 cDNA (B16-PRL3) had metastatic abilities comparable to those of Bl16F10 cells. To study the molecular mechanisms that underlie metastasis, the proteomes of the B16, B16F10, and B16-PRL3 cell lines were compared using two-dimensional differential in-gel electrophoresis. Proteins that varied significantly in levels between these cell lines were selected and identified using mass spectrometry. Interestingly, many proteins, especially those present in membrane fractions, were similarly up-or downregulated in both the Bl16F10 and B16-PRL3 cells lines compared to B16 cell lines. The list of similarly regulated proteins included heat shock protein 70, fascin-1, septin-6, ATP synthase beta subunit, and bone morphogenic protein receptor type IB. These proteins may play a causal role in PRL-3-mediated metastasis. These investigations open an avenue for the further characterization of the molecular mechanisms that underlie metastasis.