Hydrophobic interaction chromatography (HIC) has been used extensively for the separation of proteins and peptides by elution using a descending salt gradient, with and without the use of detergents or denaturing agents. In this paper we compare different hydrophobic interaction chromatographic media for the separation of multiple forms of hexokinase from rabbit reticulocytes. Among the different hydrophobic chromatographic media tested (Toyopearl Phenyl 650S, Ether 650S and Butyl 650S) Toyopearl Phenyl 650S offered the best separation of multiple forms of hexokinase, probably due to its intermediate hydrophobicity. In order to establish the optimal experimental conditions, we evaluated the effects of different salts, and the results obtained demonstrated that among the antichaotropic salts, ammonium sulphate is the most suitable for the separation of hexokinase sub-types. The sample loading capacity of the three Toyopearl supports was investigated and the recovery of enzymatic activity obtained ranged from 60% to 90%, depending on the different salts and hydrophobic media used. The chromatographic profiles of hexokinase activity from various mammalian and fungal tissues also demonstrate that Toyopearl Phenyl 650S can be successfully employed for the separation of multiple forms of enzymes from different biological sources.