Summary The amidation assay described in this chapter is a rapid and straightforward method for the estimation of amidating activity. Such a method is useful for the screening of tissues for amidating activity that may suggest the presence of novel amidated bioactive peptides, as may occur in the atrium of the heart. Studies of the distribution of PAM activity may also reveal the subcellular sites of peptide amidation; in the sheep hypothalamopituitary system, levels of amidating activity were much greater at the site of peptide synthesis than at the site of storage and release. Finally, amidation assays can also be used to study the effects of endocrine or other interventions (such as chronic glucocorticoid treatment or acute hypoglycemia) on the levels of amidating activity. Regulation of amidating activity is likely to parallel that of the peptide products, and identification of PAM regulation in tissues such as the heart may suggest physiological roles for as yet unidentified amidated peptides.