Photon penetration into living tissue is highly dependent on the absorption and scattering properties of tissue components. The near-infrared region of the spectrum offers certain advantages for photon penetration, and both organic and inorganic fluorescence contrast agents are now available for chemical conjugation to targeting molecules. This review focuses on those parameters that affect image signal and background during in vivo imaging with near-infrared light and exogenous contrast agents. Recent examples of in vivo near-infrared fluorescence imaging of animals and humans are presented, including imaging of normal and diseased vasculature, tissue perfusion, protease activity, hydroxyapatite and cancer.