Human pituitary glutaminyl cyclase (hQC) was expressed in Drosophila S2 cells under the control of an inducible metallothionene promoter and fused to the Drosophila immunoglobulin-binding protein signal sequence to enable secretion into the culture media. Expression levels reached 50 microg/mL culture media after 7 days of induction. The enzyme was purified to homogeneity directly from culture media by affinity chromatography on Reactive Blue 4-agarose using a step pH elution. The identity of the expressed protein was confirmed by peptide mass mapping and Western blotting. Glutaminyl cyclase was expressed as a fully active 37 kDa enzyme with kcat/Km values of 14.3, 9.3, and 2.4 mM(-1)s(-1) for the substrates Gln-Gln, Gln-NH(2), and Gln-t-butyl ester, respectively. The two cysteines were disulfide bonded, and the lone predicted glycosylation site, asparagine 49, was shown by both enzymatic deglycosylation of the expressed enzyme and site-directed mutagenesis to be glycosylated.