An accidental intra- and interspecies transmission of scrapie occurred in Italy in 1997 and 1998 following exposure to a vaccine against Mycoplasma agalactiae. PrP(Sc) in affected sheep and goats, collected from a single flock exposed to vaccination 2 years earlier, was molecularly typed. In five animals with iatrogenic scrapie, a PrP(Sc) type with a 20 kDa core fragment was found in all areas of the brain investigated. In three sheep and one goat, this isoform co-occurred with a fully glycosylated isoform that had a protease-resistant backbone of 17 kDa, whereas in two sheep and four goats, the two PrP(Sc) types were detected in different regions of the brain. In sheep with natural field scrapie, a PrP(Sc) type with physico-chemical properties indistinguishable from the 20 kDa isoform was found. The present results suggest the co-presence of two prion strains in mammary gland and brain homogenates used for vaccination.