Amyloid fibrils are aggregated and precipitated forms of protein in which the protein exists in highly ordered, long, unbranching threadlike formations that are stable and resistant to degradation by proteases. Fibril formation is an ordered process that typically involves the unfolding of a protein to partially folded states that subsequently interact and aggregate through a nucleation-dependent mechanism. Here we report on studies investigating the molecular basis of the inherent propensity of the milk protein, -casein, to form amyloid fibrils. Using reduced and carboxymethylated -casein (RCM-CN), we show that fibril formation is accompanied by a characteristic increase in thioflavin T fluorescence intensity, solution turbidity, and ␤-sheet content of the protein. However, the lag phase of RCM-CN fibril formation is independent of protein concentration, and the rate of fibril formation does not increase upon the addition of seeds (preformed fibrils). Therefore, its mechanism of fibril formation differs from the archetypal nucleationdependent aggregation mechanism. By digestion with trypsin or proteinase K and identification by mass spectrometry, we have determined that the region from Tyr 25 to Lys 86 is incorporated into the core of the fibrils. We suggest that this region, which is predicted to be aggregation-prone, accounts for the amyloidogenic nature of -casein. Based on these data, we propose that fibril formation by RCM-CN occurs through a novel mechanism whereby the rate-limiting step is the dissociation of an amyloidogenic precursor from an oligomeric state rather than the formation of stable nuclei, as has been described for most other fibril-forming systems.