Modified amyloid β (Aβ) peptides represent major constituents of the amyloid deposits in Alzheimer’s disease and Down’s syndrome. In particular, N-terminal pyroglutamate (pGlu) following truncation renders Aβ more stable, increases hydrophobicity and the aggregation velocity. Recent evidence based on in vitro studies suggests that the cyclization of glutamic acid, leading to pGlu-Aβ, is catalyzed by the enzyme glutaminyl cyclase (QC) following limited proteolysis of Aβ at the N-terminus. Here, we studied the pGlu-formation by rat QC in vitro as well as after microinjection of Aβ(1–40) and Aβ(3–40) into the rat cortex in vivo/in situ with and without pharmacological QC inhibition. Significant pGlu-Aβ formation was observed following injection of Aβ(3–40) after 24 h, indicating a catalyzed process. The generation of pGlu-Aβ from Aβ(3–40) was significantly inhibited by intracortical microinjection of a QC inhibitor. The study provides first evidence that generation of pGlu-Aβ is a QC-catalyzed process in vivo. The approach per se offers a strategy for a rapid evaluation of compounds targeting a reduction of pGlu formation at the N-terminus of amyloid peptides.