Real-time detection of targeted contrast agent binding is challenging due to background signal from unbound agent. 19F diffusion weighted MR spectroscopy (DWS) could selectively detect binding of angiogenesis-targeted perfluorocarbon nanoparticles in vivo. Transgenic K14-HPV16 mice with epidermal squamous carcinomas exhibiting up-regulated neovasculature were used, with nontransgenic littermates as controls. Mice were treated with αvβ3-integrin targeted perfluorocarbon nanoparticles. 19F DWS (b-values from 0 to 16,000 s/mm2) was performed on mouse ears in vivo at 11.74 Tesla. Progressive decay of 19F signal with increased diffusion weighting at low b-values (< 1500 s/mm2) was observed in ears of both K14-HPV16 and control mice, demonstrating suppression of background 19F signal from unbound nanoparticles in the blood. Much of the 19F signal from ears of K14-HPV16 mice persisted at high b-values, indicating a stationary signal source, reflecting abundant nanoparticle binding to angiogenesis. 19F signal in controls decayed completely at high b-values (> 1500 s/mm2), reflecting a moving signal source due to absence of angiogenesis (no binding sites). Estimated ADCs of nanoparticles in K14-HPV16 and control mice were 33.1 ± 12.9 μm2/s and 19563 ± 5858 μm2/s (p < 0.01). In vivo 19F DWS can be used for specific detection of bound perfluorocarbon nanoparticles by selectively suppressing background 19F signal from nanoparticles flowing in blood. Magn Reson Med 60:1232–1236, 2008. © 2008 Wiley-Liss, Inc.