The binding mechanism of Congo red (CR) to Alzheimer's disease (AD) amyloid fibrils (A beta) in terms of binding affinity and number of sites was quantitated from absorption spectroscopy (at 200-700 nm) by measuring the concentration of CR bound (CR-B) to AD A beta assemblies as a function of CR concentration and pH in 80% ethanol. The rationale for the use of this high concentration of ethanol derives from its use in histological screens for amyloid in tissue sections. Moreover, free CR can be separated from bound CR by filtration in ethanolic but not aqueous medium. The A beta analogs studied here included: (1) peptides having different lengths: A beta1-40, A beta11-28, A beta13-28, A beta19-28, A beta11-25; (2) wildtype, control sequences of A beta1-40 and sequences having different natural amino acid substitutions: primate Pr1-40, rodent Ro1-40, hereditary cerebral haemorrhage with amyloidosis, Dutch type (HCHWA-D) Du1-40, primate reverse sequence Pr40-1; and (3) A beta11-25 sequences having different substitutions: H13D, H14D, and D23K. Negative-staining showed that A beta1-40 fibrils in buffer were indistinguishable from those in buffered ethanolic medium. For all amyloid analogs except A beta19-28, which has no histidine residues and showed no CR binding over the entire pH range 4.0-9.5, CR-B decreased as a function of increasing pH. The decrease was steepest at about pH 5 and became zero above pH 7. For analogs having the same number of histidines, CR-B fell on the same binding curve, indicating that histidine residues are the likely binding sites for CR in this medium. The pH titration of the binding was parameterized by the stoichiometry of dye to the sites, the number of histidines per molecule, the binding dissociation constant Kd, and the apparent proton dissociation constant pK of the histidine; and the calculated pH-titration curves were found to fit the observed ones. For the peptides having 1-3 histidines the average pK was 5.0-5.5, which was similar to the expected pK of histidine in low dielectric medium (80% ethanol), and the Kd's were 2.8-5.9 microM. That histidine residues underlie CR binding in A beta amyloid is consistent with previous findings that A beta peptides sediment as fibrillar assemblies at pH-3-7 and bind Congo red over the same pH range in aqueous medium. Further, the conformation near the binding motif His13-His14-Gln15-Lys16 in A beta assemblies is not greatly altered in 80% ethanol.