Highly selective monoclonal antibodies to the peptide hormone human amylin have been produced and characterized. These antibodies are produced by hybridomas resulting from the fusions of BALB/c-derived myelomas and splenocytes from either inbred or outbred mouse strains. Certain of these antibodies recognize epitopes at the amino-terminus or the amidated carboxy-terminus, as well as conformational epitopes within the central region of the 37 amino acid peptide. Several of these antibodies show less than 0.1% cross-reactivity with related peptide hormones such as calcitonin and calcitonin gene-related peptide (CGRP) and have apparent affinities in the low nanomolar range. Antibody pairs were selected for use in two-site assays for the direct measurement of endogenous amylin and the synthetic human amylin analogue, pramlintide (25, 28, 29 tripro-human amylin), which is presently under clinical investigation for improving glucose control in patients with both Type I and Type II diabetes treated with insulin.