The aggregation process of beta-lactoglobulin (beta-Ig) from 0 min to 20 h was studied using atomic force microscopy (AFM), scanning transmission electron microscopy (STEM), sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and in situ attenuated total reflectance-Fourier transform infrared spectroscopy (ATR-FTIR). Fibril assembly was monitored in real time using AFM up to 20 h. From 0 to 85 min, beta-Ig monomers deformed and expanded with some aggregation. After 85 min, fibrillar structures were formed, exceeding 10 mu m in length. Fibrillar structures were confirmed by STEM. Secondary structural changes occurring during fibril formation were monitored by ATR-FTIR at 80 degrees C and indicated a decrease in alpha-helix content and an increase in beta-sheet content. SDS-PAGE indicated that fibrils were composed of polypeptides and not intact monomers. In this study, beta-Ig and whey protein isolate (WPI)-derived fibrils, including some double helices, in water were observed by AFM under ambient conditions and in their native aqueous environment.