Previous studies identified two mammalian prion protein (PrP) polybasic domains that bind the disease-associated conformer PrP(Sc), suggesting that these domains of cellular prion protein (PrP(C)) serve as docking sites for PrP(Sc) during prion propagation. To examine the role of polybasic domains in the context of full-length PrP(C), we used prion proteins lacking one or both polybasic domains expressed from Chinese hamster ovary (CHO) cells as substrates in serial protein misfolding cyclic amplification (sPMCA) reactions. After ∼5 rounds of sPMCA, PrP(Sc) molecules lacking the central polybasic domain (ΔC) were formed. Surprisingly, in contrast to wild-type prions, ΔC-PrP(Sc) prions could bind to and induce quantitative conversion of all the polybasic domain mutant substrates into PrP(Sc) molecules. Remarkably, ΔC-PrP(Sc) and other polybasic domain PrP(Sc) molecules displayed diminished or absent biological infectivity relative to wild-type PrP(Sc), despite their ability to seed sPMCA reactions of normal mouse brain homogenate. Thus, ΔC-PrP(Sc) prions interact with PrP(C) molecules through a novel interaction mechanism, yielding an expanded substrate range and highly efficient PrP(Sc) propagation. Furthermore, polybasic domain deficient PrP(Sc) molecules provide the first example of dissociation between normal brain homogenate sPMCA seeding ability from biological prion infectivity. These results suggest that the propagation of PrP(Sc) molecules may not depend on a single stereotypic mechanism, but that normal PrP(C)/PrP(Sc) interaction through polybasic domains may be required to generate prion infectivity.