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Neuroserpin is a member of the serine proteinase inhibitor superfamily. It can undergo a conformational transition to form polymers that are associated with the dementia familial encephalopathy with neuroserpin inclusion bodies and the wild-type protein can inhibit the toxicity of amyloid-beta peptides in Alzheimer's disease. We have used a single molecule fluorescence method, two color coincidence detection, to determine the rate-limiting steps of the early stages of the polymerization of fluorophore-labeled neuroserpin and have assessed how this process is altered in the presence of A beta(1-40.) Our data show that neuroserpin polymerization proceeds first by the unimolecular formation of an active monomer, followed by competing processes of both polymerization and formation of a latent monomer from the activated species. These data are not in keeping with the recently proposed domain swap model of polymer formation in which the latent species and activated monomer are likely to be formed by competing pathways directly from the unactivated monomeric serpin. Moreover, the A beta(1-40) peptide forms a weak complex with neuroserpin (dissociation constant of 10 +/- 5 nM) that increases the amount of active monomer thereby increasing the rate of polymerization. The A beta(1-40) is displaced from the complex so that it acts as a catalyst and is not incorporated into neuroserpin polymers.