Apolipoprotein E is immunochemically localized to amyloid plaque in Alzheimer's brains, and the allelic distribution of ApoE in individuals is associated with a disposition toward Alzheimer's disease. We show here that all three ApoE isotypes exhibit a strong and specific ability to inhibit both nucleation and seeding of fibril formation by the Aβ peptide in vitro. Aβ(1−40) depleted of aggregates requires long incubation times before the onset of fibril formation, but addition of very low levels of Aβ fibrils to such reactions is sufficient to reduce or eliminate this lag time. ApoE added to such seeded reactions extends the lag time in a dose-dependent manner, so that higher levels of seeding require higher levels of ApoE to achieve a given delay time to reaction onset. This effect is observed with all three isotypes produced in Escherichia coli, as well as with plasma-derived ApoE and the N-terminal domain of ApoE3 produced in E. coli. In contrast, bovine serum albumin and the four-helix bundle protein interleukin-4 are poor inhibitors of seeding. ApoE3 can also inhibit fibril formation by Aβ(1−42). The three full-length isotypes of ApoE produced in E. coli are equipotent at inhibition. It is therefore possible that the genetics of ApoE and AD may fundamentally depend on the ability of ApoE to inhibit seeding but that the trends in the genetics must be related to something other than the specific activities of the native ApoE isoforms used in these studies. The data show ApoE to be the first member of a new class of fibril formation inhibitor that acts by blocking the seeding of fibril growth.