Hypoxia is a critical event in tumor progression and angiogenesis. Hypoxia can be detected noninvasively by a novel spectroscopic photoacoustic tomography technology (SPAT) and this finding is supported by our molecular biology investigation aimed to elucidate the etiopathogenesis of SPAT detected hypoxia and angiogenesis. The present study provides an integrated approach to define oxygen status (hypoxia) of intracranial tumor xenografts using spectroscopic photoacoustic tomography. Brain tumors can be identified based on their distorted vascular architecture and oxygen saturation (SO2) images. Noninvasive in vivo tumor oxygenation imaging using SPAT is based on the spectroscopic absorption differences between oxyhemoglobin (O2Hb) and deoxyhemoblobin (HHb). Sprague-Dawley rats inoculated intracranially with ENU1564, a carcinogen-induced rat mammary adenocarcinoma cell line, were imaged with SPAT three weeks post inoculation. Proteins important for tumor angiogenesis and invasion were detected in hypoxic brain foci identified by SPAT and were elevated compared with control brain. Immunohistochemistry, Western blotting, and semi-quantitative RT-PCR showed that HIF-1 alpha, VEGF-A, and VEGFR2 (Flk-1) protein and mRNA expression levels were significantly higher (P < 0.05) in brain tumor tissues compared to normal brain. Gelatin zymography and RT-PCR demonstrated the upregulation of MMP-9 in tumor foci compared with brain control. Together these results suggest the critical role of hypoxia in driving tumor angiogenesis and invasion through upregulation of target genes important for these functions. Moreover this report validates our hypothesis that a novel noninvasive technology (SPAT) developed in our laboratory is suitable for detection of tumors, hypoxia, and angiogenesis.