PURPOSE: Molecular imaging of alphavbeta3 integrin has exhibited real potential to guide the appropriate use of anti-angiogenic therapies. However, an incomplete understanding of the factors that influence binding of alphavbeta3 integrin-specific radiotracers currently limits their use for assessing response to therapy in cancer patients. This study identifies two fundamental factors that modulate uptake of these radiotracers. Procedures Experiments were performed in prostate cancer (PC3) and glioblastoma (U87MG) cells, which differentially express alphavbeta3 integrin. alphavbeta3 integrin-specific radiotracers were used to investigate the effect of manipulating alphavbeta3 integrin expression or activation in cellular binding assays. beta3 integrin and alphavbeta3 integrin expression were measured by western blotting and flow cytometry, respectively. The effect of select pharmacological inhibitors on alphavbeta3 integrin activation and expression was also determined. RESULTS: Radiotracer binding was proportional to alphavbeta3 integrin expression when it was decreased (beta3 knock-down cells) or increased, either using pharmacological inhibitors of cell signalling or by culturing cells for different times. Studies with both small molecule and arginine-glycine-aspartic acid (RGD)-based radiotracers revealed increased radiotracer binding after activation of alphavbeta3 integrin with Mn(2+) or talin head domain. Moreover, inhibition of fundamental signalling pathways (mitogen-activated protein kinase kinase (MEK), Src and VEGFR2) decreased radiotracer binding, reflecting reduced alphavbeta3 integrin activity. CONCLUSION: Binding of small molecule ligands and radiolabelled RGD peptides is modulated by expression and activation status of alphavbeta3 integrin. alphavbeta3 integrin-specific radiotracers can provide otherwise inaccessible information of the effect of signalling pathways on alphavbeta3 integrin. This has significant implications for assessing response to anti-angiogenic therapies in clinical studies.