Publisher Summary This chapter deals with the discovery of the Hexosaminidase Isoenzymes. The definition of the clinical symptoms and the realization that this was a familial disorder, long preceded the laboratory analytical technology that was necessary for a precise identification of the biochemical lesion. As precise assay of enzyme activity on these hydrophobic or amphipathic natural substrates was an exacting task necessitating the preparation and use of radio-labelled glycolipids, many workers elected to study the enzymology of mammalian glycosidases using methods based on synthetic water-soluble substrates. This chapter illustrates how skill and perseverance with these natural substrates led to the discovery of such an activating factor and also reveals, at a very early stage, the remarkable degree of genetic heterogeneity that could arise in what turned out to be a family of defects. The effect could be attributed to Hexosaminidase A being a multisialated version of Hexosaminidase B. The ultimate reduction of electrophoretic mobility to that of Hexosaminidase B and the transient appearance of a B-like species during the heat denaturation of A, supported a very close structural relationship.