In order to better understand the molecular and cellular determinants of tumor cell intravasation, our laboratory has generated a pair of congenic human HT-1080 fibrosarcoma variants (i.e. HT-hi/diss and HT-lo/diss) differing 50-100-fold in their ability to intravasate and disseminate. To investigate the molecular differences underlying the distinct dissemination capacities of these HT-1080 variants, we performed a comparative analysis of the cell surface proteomes of HT-hi/diss and HT-lo/diss. Cell membrane proteins were enriched by biotinylation and avidin precipitation and analyzed by tandem mass spectrometry employing multidimensional protein identification technology. By this approach, 47 cell surface-associated molecules were identified as differentially expressed between the HT-1080 intravasation variants. From these candidates, four targets (i.e. TIMP-2, NCAM-1, JAM-C, and tissue factor (TF)) were selected for further biochemical validation and in vivo functional verification. Western blot analysis of the cell surface enriched fractions confirmed the proteomic array data, demonstrating that, in vitro, TIMP-2 protein was increased in the HT-lo/diss variant, whereas NCAM-1, JAM-C, and TF levels were increased in the HT-hi/diss variant. Corresponding in vivo differences in levels of TIMP-2, JAM-C, and TF were demonstrated in primary tumors grown in the chick embryo. Finally, functional inhibition of one selected protein (i.e. TF) by small interfering RNA silencing or ligation with a function-blocking antibody significantly reduced HT-hi/diss intravasation, thus clearly implicating TF in the early steps of tumor cell dissemination. Overall, our cell surface proteomic analysis provides a powerful tool for identification of specific cell membrane molecules that contribute functionally to intravasation and metastasis in vivo.