Metachromatic leukodystrophy (MLD) is a neurologically devastating autosomal recessive disorder in humans associated with deficient arylsulfatase A activity. However, clinically normal individuals described as being pseudo-arylsulfatase-A deficient also demonstrate the same deficiency. Genotypically, they may be homozygous for the pseudodeficiency mutation (associated with 2 A-->G transitions in the cDNA of arylsulfatase A) or heterozygous with one pseudodeficiency and one MLD allele. Using as examples 2 families in which the pseudo deficiency condition occurs either independently or together with MLD, we demonstrate the utility of a proposed diagnostic protocol to provide complete genotype identification of individuals suffering from arylsulfatase A deficiency. Patient fibroblasts are extracted for DNA and a cytoplasmic fraction, which is used for arylsulfatase A enzyme assay. This will identify an arylsulfatase A-deficient group, which is further analyzed electrophoretically. Cells from the clinically affected patients with MLD are completely deficient in arylsulfatase A activity, whereas those from the pseudodeficient individuals demonstrate a characteristic residual arylsulfatase A activity detectable only after electrophoresis. Within this pseudodeficient group, gene amplification of DNA specific for the A-->G mutations will distinguish between those who are homozygous for the pseudodeficiency allele and those who are compound heterozygous for the pseudodeficiency and MLD alleles. This protocol of complete genotype identification requires only about 10(6) fibroblasts (1 x 100 mm dish) and 2 days to complete. Such variant-specific genotype identification increases accuracy and prognostic value of the diagnosis. It will likely become the preferred choice for diagnosis of genetic disease in the future as more variant-specific mutations are identified at the molecular level.