Objective: Annexin V, a human protein with a high affinity for phosphatidylserine (PS), has been labeled to detect apoptosis in vivo.Vascular endothelial groth factor (VEGF) plays a pivotal role in tumor neovascularization and also can protect tumor cell from apoptosis in association with a significant up-regulation in the anti-apoptotic protein Bcl-2.To determine the effectiveness of imaging with 99mTc directly labeled recombinant human Annexin V (99mTc-rh-Annexin V) as a reflection of the degree of apoptosis in tumor, we studied mice with an engrafted hepatoma, at the same time, investigated the relationship of 99mTc-rh-annexin V distribution and the expression of VEGF and Bcl-2 early after a single dose of radiotherapy.Methods: Eight days after being inoculated with allogenic hepatoma cells(Hca-F25) into the subcutaneouly of the right axillary fossa, the mice (purebred 615) were randomly divided into tow groups (group A was control group, n=9; group B was treated group, n=10).Group B was randomly divided into three groups and received different dose of radiotherapy (group B 1, n=4,2Gy; group B2, n=3, 5Gy; group B3, n=3, 10Gy).One hour after radiotherapy, group A and group B received 99mTc-rh-annexinV(3.7MBq/0.5μg/per mouse) injection intravenously, and 4 hours later, the animals were imaged and scarified.The accumulation of 99mTc-rh-AnnexinV in tumor was expressed as the tumor-to-normal tissue (T/NT).Tumor cell apoptosis was examined by TUNEL methods, and the expression of VEGF and Bcl-2 in tumor were determined with immunohistochemical methods.Results: Radiotherapy significantly increased the tumor uptake of 99mTc-rh-Annexin V and the positive number of TUNEL, but the expression of Bcl-2 was significantly high in group A than in group B(P<0.01).There were no statistical significance between group A and group B in expression of VEGF.The T/NT in tumor correlated well with the positive number of TUNEL in group A and group B (r=0.785, r=0.812).The expression of Bcl-2 correlated negatively with the T/NT and the positive number of TUNEL in group A and group B, but no statistical significance (P > 0.05) The expression of VEGF correlated negatively with the distribution of 99mTc-rh-Annexin V and positively with expression of Bcl-2 in group A (r=-0.794, r =-0.736),but it had no relationship with the other two in group B.Conclusions: The distribution of 99Tcm-rh-AnnexinV in tumor could reflected the degree of apoptosis early after a single dose of radiotherapy more truly than the expression of VEGF, and maybe the anti-apoptosis function expression of VEGF needed high level expression of Bcl-2 in tumor.