Integrin-associated protein (IAP) is a receptor for the carboxyl-terminal "cell-binding domain" (CBD) of thrombospondin 1 (TS1). IAP associates with alpha v beta 3 integrin and mAbs against IAP inhibit certain integrin functions. Here we examine the effects of the TS1 CBD and 4N1K (KRFYVVMWKK), a cell-binding peptide derived from it, on the adhesion and spreading on vitronectin (VN) of C32 human melanoma cells which express IAP, alpha v beta 3, and alpha v beta 5. Cells adhere to VN at low surface densities via alpha v beta 5 and spread very slowly while adhesion to higher density VN involves both alpha v beta 5 and alpha v beta 3 and results in rapid spreading. Spreading of the cells, but not adhesion, on sparse VN coatings is markedly enhanced by the presence of soluble TS1, the recombinant CBD and 4N1K, but not the "mutant" peptide 4NGG, KRFYGGMWKK, which fails to bind IAP. This enhanced spreading is completely blocked by mAb LM609 against alpha v beta 3 and the anti-IAP mAb B6H12. Correlated with this enhanced spreading is increased tyrosine phosphorylation of focal adhesion kinase (FAK), paxillin, and a protein of ca. 90 kD. The enhanced spreading induced by TS1 and 4N1K and the constitutive spreading on higher density VN are both blocked by calphostin C (100 nM), wortmannin (10 nM), and tyrosine kinase inhibitors. In contrast, pertussis toxin specifically blocks only the TS1 stimulated spreading on low density VN, indicating that IAP exerts its effects on signal transduction via a heterotrimeric Gi protein acting upstream of a common cell spreading pathway which includes PI-3 kinase, PKC, and tyrosine kinases.