Aspartylglucosaminuria (AGU) is a lysosomal storage disease caused by deficient activity of aspartylglucosaminidase (AGA). We report here a T214C mutation leading to a Ser72Pro substitution in four Arab families. This is the first naturally occurring AGU mutation involving an active-site amino acid of this recently crystallized hydrolase and it seems to represent the second most common AGU mutation worldwide. The intracellular consequences of the Ser72Pro mutation were analyzed by transient expression in COS-1 cells and we were able to demonstrate that this active-site mutation most probably does not destroy the enzyme activity per se, but specifically prevents the proteolytic activation cleavage of AGA in the endoplasmic reticulum (ER). The mutant enzyme is, however, folded correctly enough to allow mannose-6-phosphorylation and targeting to lysosomes. The overexpressed mutant enzyme remained inactive intracellularly, but the secreted mutant precursor was proteolytically activated extracellularly, resulting in a similar subunit composition to that in the wild-type AGA in the ER. The partially activated mutant enzyme was endocytosed further by the recipient cells. These data demonstrate that the proteolytic activation of AGA can also occur extracellularly and suggest that the driving mechanism of AGA precursor cleavage is autocatalytic.