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The fluorescent dye 1,6-diphenyl-1,3,5-hexatriene (DPH) is widely used as a probe of membrane order. We show that DPH also interacts with amyloid fibrils formed by human amylin (h-amylin, also known as islet amyloid polypeptide) in solution, and this results in a 100-fold increase in DPH fluorescence for a sample of 20 μM h-amylin and 0.25 μM DPH. No increase in DPH fluorescence is observed with the non-amyloidogenic rat amylin or with freshly dissolved, nonfibrillar h-amylin. The time course of amyloid formation by amylin was followed by monitoring the fluorescence of added DPH as a function of time and was similar to that monitored by the standard fluorescent probe thioflavin-T. The inclusion of DPH in the buffer did not perturb the time course of amyloid formation under the conditions examined, and the time course was independent of the range of DPH concentrations tested (0.25-5 μM). The maximum final fluorescence intensity is observed at substoichiometric ratios of DPH to amylin. No significant increase in fluorescence was observed during the lag phase of amyloid formation, and the implications for the structure of amylin prefibril oligomers are discussed. h-Amylin contains three aromatic residues. A triple aromatic to leucine mutant forms amyloid, and DPH binds to the resulting fibrils, indicating that interactions with aromatic side chains are not required for DPH-amylin amyloid interactions. DPH may be especially useful for studies of mutant amylins and other polypeptides in which changes in charged residues might complicate interpretation of thioflavin-T fluorescence.