Molecular imaging is a newly emerging field in which the modern tools of molecular and cell biology have been married to state-of-the-art technologies for noninvasive imaging. The study of molecular imaging will lead to better methods for understanding biological processes as well as diagnosing and managing disease. Here we present noninvasive in vivo spectroscopic photoacoustic tomography (PAT)-based molecular imaging of αvβ3 integrin in a nude mouse U87 brain tumor. PAT combines high optical absorption contrast and high ultrasonic resolution by employing short laser pulses to generate acoustic waves in biological tissues through thermoelastic expansion. Spectroscopic PAT-based molecular imaging offers the separation of the contributions from different absorbers based on the differences in optical absorption spectra among those absorbers. In our case, in the near infrared (NIR) range, oxy-heamoglobin (O2Hb), deoxy-heamoglobin (HHb) and the injected αvβ3-targeted peptide-ICG conjugated NIR fluorescent contrast agent are the three main absorbers. Therefore, with the excitation by multiple wavelength laser pulses, spectroscopic PAT-based molecular imaging not only provides the level of the contrast agent accumulation in the U87 glioblastoma tumor, which is related to the metabolism and angiogenesis of the tumor, but also offers the information on tumor angiogenesis and tumor hypoxia.© (2006) COPYRIGHT SPIE--The International Society for Optical Engineering. Downloading of the abstract is permitted for personal use only.