EphB receptors and their ephrinB ligands play a key role in the formation of a regular vascular system. Recent studies have also shown the involvement of Eph/ephrin interactions in malignant tumor progression and angiogenesis. We have generated soluble monomeric EphB4 (sEphB4)-expressing A375 melanoma cells to study the effect of dominant negatively acting sEphB4 on tumor growth and angiogenesis. Soluble EphB4-expressing A375 tumors grown subcutaneously in nude mice show dramatically reduced tumor growth compared to control tumors. The proliferative capacity of sEphB4-expressing cells in monolayer culture is not altered. Yet, sEphB4-expressing A375 cells cannot establish proper cell-cell contacts in three-dimensional spheroids. However, sEphB4 transfectants have reduced proliferation and apoptosis rates when grown in three-dimensional culture in vitro or in subcutaneous tumors in vivo. Analysis of the vascular phenotype of the tumors revealed a reduction of intratumoral microvessel density in sEphB4-expressing tumors. Corresponding to these mouse experiments, a matched pair analysis of EphB4 and ephrinB2 expression in human colon carcinomas revealed significantly upregulated levels of EphB4 expression compared to adjacent normal tissue. Taken together, the data identify dual effects of sEphB4 on the tumor and the vascular compartment that collectively inhibit tumor growth.