Syndecans are cell surface heparan sulfate proteoglycans with regulatory roles in cell adhesion, proliferation, and differentiation [Annu. Rev. Biochem. 68 (1999) 729]. While the syndecan heparan sulfate chains are essential for matrix binding, less is known about the signaling role of their core proteins. To mimic syndecan-specific adhesion, MDA-MB-231 mammary carcinoma cells were plated on antibodies against syndecan-4 or syndecan-1. While cells adherent via syndecan-4 spread, cells adherent via syndecan-1 do not. However, cells adherent via syndecan-1 can be induced to spread by Mn(2+), suggesting that activation of a beta(1) or beta(3) integrin partner is required. Surprisingly, pretreatment of cells with a function-activating beta(1) antibody does not induce spreading, whereas function-blocking beta(1) integrin antibodies do, suggesting involvement of a beta(1)-to-beta(3) integrin cross-talk. Indeed, blockade of beta(1) integrin activation induces alpha(v)beta(3) integrin activation detectable by soluble fibrinogen binding. Spreading in response to syndecan-1 is independent of integrin-ligand binding. Furthermore, competition with soluble murine syndecan-1 ectodomain, which does not disrupt cell adhesion, nonetheless blocks the spreading mechanism. These data suggest that the ectodomain of the syndecan-1 core protein directly participates in the formation of a signaling complex that signals in cooperation with alpha(v)beta(3) integrins; signaling via this complex is negatively regulated by beta(1) integrins.