Insulin-like growth factor-1 (IGF-I) can bind to the extracellular matrix protein vitronectin (VN) through the involvement of IGF-binding proteins-2, -3, -4 and -5. Since IGF-I and VN have established roles in tumour cell dissemination, we were keen to investigate the functional consequences of the interaction of IGF-I, IGFBPs and VN in tumour cell biology. Hence, functional responses of MCF-7 breast carcinoma cells and "normal" non-tumourgenic MCF-10A mammary epithelial cells were investigated to allow side-by-side comparisons of these complexes in both cancerous and normal breast cells. We demonstrate that substrate-bound IGF-I:IGFBP:VN complexes stimulate synergistic increases in cellular migration in both cell types. Studies using IGF-I analogues determined this stimulation to be dependent upon both heterotrimeric IGF-I:IGFBP:VN complex formation and the involvement of the IGF-1R. Furthermore, the enhanced cellular migration was abolished upon incubation of MCF-7 and MCF-10A cells with function blocking antibodies directed at VN-binding integrins and the IGF-IR. Analysis of the signal transduction pathways underlying the enhanced cell migration revealed that the complexes stimulate a transient activation of the ERK/MAPK signalling pathway, while simultaneously producing a sustained activation of the PI3-K/AKT pathway. Experiments using pharmacological inhibitors of these pathways determined a requirement for PI3-K/AKT activation in the observed response. Overexpression of wild type and activated AKT further increases substrate-bound IGF-I:IGFBP:VN-stimulated migration. This study provides the first mechanistic insights into the action of IGF-I:IGFBP:VN complexes and adds further evidence to support the involvement of VN-binding integrins and their co-operativity with the IGF-IR in the promotion of tumour cell migration.