Fungal prions are protein-based genetic elements. Sup35 and Ure2p constitute the best-characterized prion proteins in the yeast Saccharomyces cerevisiae. No high-resolution molecular models of the amyloid conformations adopted by the prion domains of these proteins are available yet. A quantitative description of the kinetics and thermodynamics of their self-assembly processes might provide clues on the nature of the structural changes originating their heritable and transmissible phenotypes. Here we study the temperature dependence of Sup35 and Ure2p amyloid fibril nucleation and elongation reactions at physiological pH. Both processes follow the Arrhenius law, allowing calculation of their associated thermodynamic activation parameters. Although the Gibbs energies (ΔG) for the nucleation and elongation of both prions are similar, the enthalpic and entropic contributions to these two processes are dramatically different. In addition, the structural properties of the two types of prion fibrils exhibit different dependence on the polymerization temperature. Overall, we show here that the amyloidogenic pathways of Sup35 and Ure2p prions diverge significantly. © 2011 American Chemical Society.