Much remains to be learned about the physical relationship between infectivity and aggregated PrP, and to which extent it varies according to the prion strain. Transmission of natural sheep scrapie and BSE infectious sources to transgenic mice overexpressing the VRQ allele of ovine PrP (tg338 line) has allowed us to establish a panel of biologically cloned strains that have been shown to produce stable and clearly distinct phenotypes based on the electrophoretic profile of PrPSc, its regional distribution in the brain, and the incubation time. We have sought to further compare these strains on the basis of their physicochemical features. We first studied the individual conformational stability of four strains by measuring the resistance to denaturation by guanidine-HCl (GdnHCl). At variance with that recently reported for rodent-adapted strains, we failed to observe a straight correlation between [GdnHCl]1/2 values and incubation times. We next compared the sedimentation properties of abnormal PrP and infectivity of each strain by velocity gradient centrifugation, by using detergent-solubilised, infected brain homogenate instead of PrPres-enriched material. Gradient fractions were analysed for PrPC, PrPSc and PrPres content by ELISA and western blotting, and for infectivity by bioassay in tg338 mice. The detergent treatment conditions were optimised so as to separate most of the PrPC from the bulk of PrPres. PrPres was found to sediment in the middle region of the gradient, however the position and shape of the peak noticeably differed among the strains, reflecting PrPSc aggregates of different sizes. Examining the distribution of infectivity revealed even more striking differences. Thus, 99% of the infectivity was separated from about 90% of PrPres in the case of a fast strain, whereas in the case of BSEov agent the peaks of PrPres and infectivity were largely overlapping. From these results it was concluded that the relationship between infectivity and misfolded PrP multimers is not univocal, suggesting that the size of the most infectious particles may substantially differ among prions strains.