Purpose: Tc-99m-3P-RGD(2) is a Tc-99m-labeled dimeric cyclic RGD peptide that binds to integrin alpha(v)beta(3) with high affinity and specificity. The purpose of this study was to demonstrate the utility of Tc-99m-3P-RGD(2) SPECT/CT (single photon emission computed tomography/computed tomography) as a molecular imaging tool for noninvasive monitoring breast tumor early response to antiangiogenesis therapy with linifanib, and to illustrate its limitations in monitoring the efficacy of anti-alpha(v)beta(3) treatment. Methods: To support SPECT/CT imaging, biodistribution and therapy studies, the xenografted breast cancer model was established by subcutaneous injection of 5 x 10(6) MDA-MB-435 cells into the fat pad of each athymic nude mouse. Linifanib (ABT-869) was used as antiangiogenesis agent. The tumor volume was 180 +/- 90 mm(3) on the day (-1 day) before baseline SPECT/CT. Each animal was treated twice daily with vehicle or 12.5 mg/kg linifanib. Longitudinal Tc-99m-3P-RGD(2) SPECT/CT imaging was performed on days -1, 1, 4 and 11. Tumors were harvested at each time point for pathological analysis of hematoxylin and eosin (H&E) and immunohistochemistry (IHC). Tumor uptake of Tc-99m-3P-RGD(2) was calculated from SPECT/CT quantification. When cyclic peptide E[c(RGDfK)](2) (RGD(2)) was used as the anti-alpha(v)beta(3) agent, SPECT/CT images were obtained only at 7 and 21 days after last RGD(2) dose. Results: The tumor uptake of Tc-99m-3P-RGD(2) from SPECT/CT quantification was almost identical to that from biodistribution. There was a dramatic reduction in both %ID and %ID/cm(3) tumor uptake of Tc-99m-3P-RGD(2) during the first 24 hours of linifanib therapy. The therapeutic effect of linifanib was on both tumor cells and vasculature, as determined by IHC analysis of integrin alpha(v)beta(3) and CD31. Changes in tumor vasculature were further confirmed by pathological H& E analysis of tumor tissues. While its %ID tumor uptake increased steadily in vehicle-treated group, the %ID tumor uptake of Tc-99m-3P-RGD(2) decreased in linifanib-treated group slowly over the 11-day study period. The degree of tumor response to linifanib therapy correlated well to the integrin alpha(v)beta(3) expression levels before linifanib therapy. Conclusion: Tc-99m-3P-RGD(2) is an excellent radiotracer for monitoring integrin alpha(v)beta(3) expression during and after linifanib therapy. Tc-99m-3P-RGD(2) SPECT/CT is an useful molecular imaging tool for patient selection before antiangiogenic and anti-alpha(v)beta(3) therapy; but it would be difficult to use Tc-99m-3P-RGD(2) for accurate and noninvasive monitoring of early tumor response to anti-alpha(v)beta(3) therapy.