Tumor‐cell extravasation involves sequential adhesive interactions with vascular endothelium and the subendothelial basement membrane. We have established a 3‐dimensional model in vitro to simulate these events and to elucidate targets of the anti‐cell‐adhesive synthetic peptide RGDS. Tumor spheroids of the melanoma cell line ST‐ML‐12 served as models of tumor‐cell emboli and were transferred onto human umbilical vein endothelial cells. To imitate the vascular anatomy, the latter were grown on reconstituted basement membranes produced by dextran‐stimulated bovine corneal endothelial cells. RGDS did not affect the homotypic aggregation of the tumor cells and only minimally inhibited the attachment of the spheroids to the reconstituted vessel. A short‐term (20 min) inhibition of adhesion to denuded basement membranes was observed. The attachment was closely associated with damage to the endothelial cells by oxygen‐derived free radicals. RGDS retarded endothelial injury for up to 3 hr. The most prominent effect was observed after penetration of the endothelium. RGDS suppressed the emigration of tumor cells from the attached tumor‐cell cluster in a dose‐ and time‐dependent fashion. After 12 hr, the inhibitory effect progressively declined. This was not due to loss of activity of the peptide, indicating a resistance mechanism in the melanoma cells. On purified components of the basement membrane, RGDS effectively inhibited the initial spheroid attachment to fibronectin and collagen IV but had no effect on attachment to laminin. By contrast, subsequent migration was significantly suppressed on all substrata. Our model permits the study of dynamic cell‐cell and cell‐extracellular‐matrix interactions and indicates that RGDS might predominantly act on early tumor‐cell locomotion after penetration of the endothelium.