In most prion diseases, infectivity accumulates in lymphoreticular organs early after infection. Defects in hematopoietic compartments, such as impaired B-cell maturation, or in stromal compartments, such as abrogation of follicular dendritic cells, can delay or prevent lymphoreticular prion colonization. However, the nature of the compartment in which prion replication takes place is controversial, and it is unclear whether this compartment coincides with that expressing the normal prion protein (PrP^c). Here we studied the distribution of infectivity in splenic fractions of wild-type and fetal liver chimeric mice carrying the gene that encodes PrP^c (Prnp) solely on hematopoietic or on stromal cells. We fractionated spleens at various times after intraperitoneal challenge with prions and assayed infectivity by bioassay. Upon high-dose challenge, chimeras carrying PrP^con hematopoietic cells accumulated prions in stroma and in purified splenocytes. In contrast, after low-dose challenge ablation ofPrnp in either compartment prevented splenic accumulation of infectivity, indicating that optimal prion replication requires PrP^c expression by both stromal and hematopoietic compartments.