Fabry disease is an X-linked inborn error of glycolipid metabolism caused by a deficiency of the lysosomal enzyme alpha-galactosidase A (GalA; EC 3.2.1.22). In order to obtain large quantities of this human enzyme for physical characterization and for the development of new approaches for enzyme therapy, we constructed derivatives of the Autographa californica nuclear polyhedrosis virus that produce the human enzyme. The recombinant GalA (re-GalA) is produced at high levels, and is active with both the artificial substrate, 4-methylumbelliferyl-alpha-D-galactopyranoside, and the natural in vivo substrate, trihexosylceramide. The purified re-GalA is glycosylated and is taken up by normal and Fabry fibroblasts in cell culture. Mass spectral analysis of total monosaccharides released by hydrazinolysis indicates that it contains fucose, galactose, mannose and N-acetylglucosamine. Amino-acid sequence analysis of six proteolytic peptides corresponded to sequences predicted by the cDNA. The molecular masses of the purified enzyme, estimated by electrospray mass spectroscopy and laser desorption time-of-flight analysis are 46.85 and 46.62 kDa, respectively, approx. 10% greater than the polypeptide portion predicted by the cDNA. The recombinant enzyme retains significant catalytic activity after modification with poly(ethylene glycol), a treatment which decreases the immunogenicity and increases the circulation life of many proteins used therapeutically.