Prions are the infectious agents responsible for prion diseases, which appear to be composed exclusively by the misfolded prion protein (PrP(Sc)). The mechanism of prion transmission is unknown. In this study, we attempted to detect prions in urine of experimentally infected animals. PrP(Sc) was detected in approximately 80% of the animals studied, whereas no false positives were observed among the control animals. Semi-quantitative calculations suggest that PrP(Sc) concentration in urine is around 10-fold lower than in blood. Interestingly, PrP(Sc) present in urine maintains its infectious properties. Our data indicate that low quantities of infectious prions are excreted in the urine. These findings suggest that urine is a possible source of prion transmission.