Intravascular cancer cell adhesion plays a significant role in the metastatic process. Studies indicate that galectin-3, a member of the galectin family of soluble animal lectins, is involved in carbohydrate-mediated metastatic cell heterotypic (between carcinoma cells and endothelium) and homotypic (between carcinoma cells) adhesion via interactions with the tumor-specific Thomsen-Friedenreich glycoantigen (TFAg). We hypothesized that blocking the galectin-3 carbohydrate recognition domain with synthetic peptides would significantly reduce metastasis-associated carcinoma cell adhesion. To test this hypothesis, we identified peptide antagonists of the galectin-3 carbohydrate recognition domain using combinatorial bacteriophage display technology. The peptides bound with high affinity to purified recombinant galectin-3 protein (K(d) approximately 17-80 nM) and to cell surface galectin-3. Experiments with a series of recombinant serially truncated galectin-3 mutants indicated that the peptides bound the carbohydrate recognition domain of galectin-3. Furthermore, the peptides did not bind the carbohydrate recognition domain of other galectins and plant lectins. Synthetic galectin-3 carbohydrate recognition domain-specific peptides blocked the interaction between galectin-3 and TFAg and significantly inhibited rolling and stable heterotypic adhesion of human MDA-MB-435 breast carcinoma cells to endothelial cells under flow conditions, as well as homotypic tumor cell aggregation. These results demonstrate that carbohydrate-mediated, metastasis-associated tumor cell adhesion could be inhibited efficiently with short synthetic peptides which do not mimic naturally occurring glycoepitopes yet bind to the galectin-3 carbohydrate recognition domain with high affinity and specificity.