Arylsulfatase A (ASA) pseudodeficiency (PD) was described in clinically healthy individuals with ASA-deficient activity. To confirm that the PD individual in the present study is homozygous for the PD allele without any other mutations, direct solid-phase sequencing was done and the two A-to-G transitions--one at the third N-glycosylation site (N350S) and the other at the first polyadenylation signal (ATTAAC to AGTAAC)--were identified. No other mutations were detected in the entire coding region nor in the intron-exon boundary region of the ASA gene in the PD cells. Kinetic studies to compare the partially purified ASA from controls to that from a homozygote (PD allele) were carried out using p-nitrocatechol sulfate (p-NCS) as a substrate. The apparent Km for the control ASA was 0.6 mM and for the PD enzyme 2.0 mM (p < 0.01). The heat inactivation at 60 degrees C revealed 50% inactivation within 90 min for control ASA and 28 min for PD ASA. At 65 degrees C, the 50% inactivation was reached at 18 min for the control and at 8.5 min for the PD. These results document the decreased affinity of ASA toward p-NCS and increased heat inactivation from a PD individual. Western blot analysis following SDS-PAGE and isoelectric focusing revealed differences in both the molecular weight and the isoelectric point between the control ASA and that of the PD allele. To the best of our knowledge, this is the first report showing the altered properties of ASA from a PD homozygote.