The detection of heterozygotes for MLD based on enzyme assays of a general population is highly unreliable. Twenty-three percent of controls and ARA activities below the levels found in some obligate heterozygotes for MLD. This serious overlap problem precludes the use of ARA determinations in large screening programs to assign individuals into specific genetic categories. On the other hand, intrafamily analysis of ARA activity offers the possibility of accurately determining heterozygotes for MLD. Sixteen children of parents heterozygous for MLD had ARA activities which clearly categorized them as either homozygous affected, heterozygous, or normal. The wide range of ARA activity found in controls and heterozygotes for MLD appeared to result from the presence of multiple allelic mutations at the ARA locus. One of these mutations leads to a low ARA activity and when present in an individual who is heterozygous for MLD may lead to overlap of his total activity with that of some homozygous affected individuals. This low ARA activity mutation can be recognized by alterations in the multiple molecular forms of ARA activity separated by analytical isoelectric focusing electrophoresis.