Amyloid fibrils share various common structural features and their presence can be detected by Thioflavin T (ThT). In this paper, the binding mode of ThT to insulin amyloid fibrils was examined. Scatchard analysis and isothermal titration calorimetry (ITC) showed at least two binding site populations. The binding site population with the strongest binding was responsible for the characteristic ThT fluorescence. This binding had a capacity of about 0.1 moles of ThT bound per mole of insulin in fibril form. The binding capacity was unaffected by pH, but the affinity was lowest at low pH. Notably, presence of a third binding process prior to the other processes was suggested by ITC. Binding of ThT resulted in only minor changes in the fibril structure according to the X-ray diffraction patterns, where a slightly more dominant equatorial reflection at 16A relative to the intersheet distance of 11A was observed. No change in the interstrand distance of 4.8A was observed. On the basis of our results, we propose that ThT binds in cavities running parallel to the fibril axis, e.g., between the protofilaments forming the fibrils. Such cavities have been proposed previously in insulin fibrils and several other amyloid fibril models.