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Viral envelopes can be used as an effective vehicle to deliver imaging tracers as well as therapeutic drugs and genes. However, the current methods for in vivo tracking of viral envelopes are limited. This purpose of this study is to investigate dynamically the in vivo biodistribution of viral envelopes using positron emission tomography (PET) imaging. The hemagglutinating virus of Japan envelope (HVJ-E) was labeled with radioactive fluorine (F-18) for tracking with PET imaging. Due to the low molecular weight of F-18, the encapsulation process by HVJ-E was optimized using the cationic agent poly-L-lysine (PLL, MW 66.7 kDa) and Feridex, a magnetic resonance imaging tracer. After labeling, HVJ-Es were injected intravenously into the normal rat and followed for 2 h using high resolution PET imaging. Region of interest analysis showed a significant increase in average liver accumulation based on radioactivity as compared to all control subjects. Average brain uptake showed a significant increase in radioactivity as compared to control subjects receiving F-18-PLL complexes or F-18 alone. Control subjects showed F-18 uptake primarily in the bones. These results demonstrate a molecular imaging technique that can be used to monitor drug and gene delivery and evaluate potential targeting mechanisms.