Extract: The activity of the glycoprotein degrading lysosomal hydrolase, 4-L-aspartylglycosylamine amido hydrolase (AADGase, EC.3.5.1.26), was measured in plasma, buffy coat leukocytes, and separated lymphocytes (Ficoll separation) from 16 patients with aspartylglucosaminuria (AGU), 29 obligate heterozygotes, and 30 control subjects. In lymphocytes the AGU patients had unmeasurable or minimal AADGase activity with a mean of 3.9 U. The obligate heterozygotes showed AADGase activities ranging from 5 to 69 U with a mean of 34.1 U. Enzyme activities in the control group ranged from 91 to 243 U with a mean of 127.9 U, and were clearly separated from the values of the heterozygotes. In leukocytes the AGU patients had unmeasurable enzyme activity and obligate heterozygotes had enzyme levels closely similar to those in the lymphocytes from the same individuals. The AADGase activity in the leukocytes of the control group displayed a much wider variation than in the lymphocytes, ranging from 22 to 132 U with a mean of 70.7 U. In plasma the AGU patients had undetectable AADGase activity. The mean enzyme level of obligate heterozygotes was 72.2 U and that of control individuals 107.2 U, but the overlap between the groups was extensive. The results indicate that homozygous deficiency of AADGase, i.e., aspartylglucosaminuria, can be reliably diagnosed using plasma, leukocytes, or separated lymphocytes. For carrier detection only separated lymphocytes allow a satisfactory differentiation between heterozygous and normal individuals. A group of 31 siblings of verified AGU cases and 11 children of identified carriers, whose spouses had normal AADGase activity, were investigated using the lymphocyte assay. The observed and expected frequencies (on the basis of Mendelian probabilities) were closely similar, suggesting that the lymphocyte assay can be used reliably for carrier detection. Speculation: Demonstration of AADGase deficiency in plasma or in white blood cells offers an alternative method for the diagnosis of aspartylglucosaminuria. Large scale screening programs for AGU carrier detection with the lymphocyte enzyme assay are not considered practical and justified at present. Carrier screening among relatives of known AGU patients is, however, medically, economically, and psychologically advantageous, particularly since prenatal diagnosis and the possibility of preventive abortion can be offered to carrier-carrier matings.