We describe a method for labeling enveloped viral particles with a radiotracer, indium-111, allowing labeled viruses to be traced in vivo by nuclear imaging. After initial optimization experiments, a labeling efficiency of 83% (incorporation yield) was achieved for herpes simplex virus (HSV), resulting in a specific activity of 30 μCi/10⁹ PFU. The labeling procedure did not significantly reduce the infectivity of the labeled virus and the virus did not release any significant amounts of the radionuclide within 12 hr after labeling. Sequential imaging of animals after intravenous administration of the labeled virus showed fast accumulation in the liver and redistribution from the blood pool (immediately after injection) to liver and spleen (12–24 hr after injection). At 12 hr after injection 7% of the virus-associated ¹¹¹In had been eliminated from the body and the remaining organ distribution of the virus was as follows: spleen 28.7 ± 5.4% ID/g; liver, 26.0 ± 5.1% ID/g; kidney, 9.8 ± 3.1% ID/g; lung, 5.7 ± 1.0% ID/g; and lower amounts in other organs. Our results indicate that the described method allows qualitative and quantitative assessment of viral biodistribution in vivo by nuclear imaging.