Background: Hypoxia in tumors may be an important factor in resistance to radiotherapy and chemotherapy. It is important to assess tumor hypoxia before selection of treatment protocols. Radionuclide imaging techniques have been developed for detecting tumors hypoxia in vivo. This study compares the distribution of two previously reported hypoxia imaging tracers, (superscript 99m)Tc-HL91 and (superscript 99m)Tc-PnAO-nitroimidazole, in an experimental tumor model. Methods: Accumulation of (superscript 99m)Tc-HL91 and 99Tc-PnAO-nitroimidazole were evaluated in eight-week-old C3HIHeN mice bearing H460 human non-small cell lung cancer. This study employed biodistribution, whole-body autoradiography and radionuclide scintigraphy methods. Hypoxia modulation by hydralazine was compared for both radiotracers. Results: At 4h after injection, the distribution of (superscript 99m)Tc-HL91 follows the order: blood＞liver＞tumor＞heart＞kidney, uscle ＞ spleen ＞ lungs, whereas the distribution of (superscript 99m)Tc-PnAO-nitroimidazole follows the sequence: kidney ＞ heart ＞＞ spleen ＞ blood ＞ muscle ＞ lung ＞ tumor. Radionuclide imaging confirmed the distribution of both tracers in the liver, G-I tract and kidneys. The uptake of the tracer in the tumor was not obvious. The distribution data do not support the success of either (superscript 99m)Tc-HL91 or (superscript 99m)Tc-PnAO-nitroimidazole to image the hypoxia enhanced by hydralazine in the tumor. Conclusion: The accumulation of (superscript 99m)Tc-HL91 in tumor was higher than (superscript 99m)Tc-PnAO-nitroimidazole. However, the accumulation of both agents in tumor in scintigraphy and autoradiography were not obvious. Further study is warranted to clarify the potential use of these agents in tumor hypoxia imaging.