The purpose of this study was to validate (99m)Tc-3P-RGD(2) single-photon emission computed tomography/computed tomography (SPECT/CT) as an imaging tool to monitor α(v)β(3) expression and tumor necrosis. The animal model was established by subcutaneous injection of 5 × 10(6) U87MG cells into the shoulder flank of each mouse. Imaging was performed using the U-SPECT-II/CT scanner (Milabs, Utrecht, the Netherlands). Tumor volumes were determined, and the tumor uptake of (99m)Tc-3P-RGD(2) was calculated on the basis of SPECT/CT and compared to that from biodistribution. Immunohistochemistry was performed to determine CD31 and α(v)β(3) expression levels. We found that the tumor detection limit was ≈ 0.5 mm(3) by (99m)Tc-3P-RGD(2) SPECT/CT. The tumor uptake of (99m)Tc-3P-RGD(2) from SPECT/CT was almost identical to that from biodistribution. The α(v)β(3) was expressed mainly on blood vessels for the tumors of 0.2 to 0.5 cm(3). In larger tumors, tumor α(v)β(3) expression increased due to more contribution from glioma cells. When tumors were > 0.5 cm(3), the %ID/cm(3) uptake of (99m)Tc-3P-RGD(2) decreased because of necrosis. The overall relationship between the tumor size and %ID of (99m)Tc-3P-RGD(2) was modeled as a quadratic polynomial fitting curve, with R(2) being > .95. (99m)Tc-3P-RGD(2) SPECT/CT is excellent for monitoring α(v)β(3) expression and tumor necrosis during tumor growth and may become a screening tool for patient selection before anti-α(v)β(3) therapy.