Hydrophobic chromatography was investigated as a purification procedure for human liver hexosaminidases. Both phenyl-Sepharose and valine-Sepharose have a high binding capacity of hexosaminidases. A degree of resolution between the A and B ioszymes is achieved with phenyl-Sepharose. Both hydrophobic supports must be used close to their capacity in order to recover the applied enzyme. Two purification procedures for human liver hexosaminidase B were employed, which resulted in recoveries of approximately 48 and 24% with final specific activities of 33,400 and 4840 nmole/min.mg, respectively.