Prion pathogenesis following oral exposure is thought to involve gut-associated lymphatic tissue, which includes Peyer's patches (PPs) and M cells. Recruitment of activated B lymphocytes to PPs requires α₄β₇ integrin; PPs of β7^−/− mice are normal in number but are atrophic and almost entirely devoid of B cells. Here we report that minimal infectious dose and disease incubation after oral exposure to logarithmic dilutions of prion inoculum were similar in β7^−/− and wild-type mice, and PPs of both β7^−/− and wild-type mice contained 3–4 log LD₅₀/g prion infectivity ≥125 days after challenge. Despite marked reduction of B cells, M cells were present in β7^−/− mice. In contrast, mice deficient in both tumor necrosis factor and lymphotoxin-α (TNFα^−/− × LTα^−/−) or in lymphocytes (RAG-1^−/−, μMT), in which numbers of PPs are reduced in number, were highly resistant to oral challenge, and their intestines were virtually devoid of prion infectivity at all times after challenge. Therefore, lymphoreticular requirements for enteric and for intraperitoneal uptake of prions differ from each other. Although susceptibility to prion infection following oral challenge correlates with the number of PPs, it is remarkably independent of the number of PP-associated lymphocytes.