The chromogenic substrate, N‐acetyl‐DL‐phenylalanine‐β‐naphthyl ester (APNE), used in trypsin or chymotrypsin studies, has been employed to identify neutral peptide hydrolases in barley by immunoelectrophoretic analysis. Enzyme activity was observed in proximal (embryo part) and distal (endosperm part) portions of barley grains before and after germination. The proximal portion contained all detectable activity in the ungerminated seed. This enzyme migrates strongly towards the anode. During germination a new enzyme is formed which hydrolyzes the same substrate but is electrophoretically different from the enzyme found in dormant grains (it is more neutral or cathodic). The newly‐formed enzyme appears in the proximal part of the seed after 34 hours of germination and in the distal part after 96 hours. This enzyme does not appear to be a carboxypeptidase but may be related to one (or more) of the reported barley ami