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During axenic growth, microplasmodia of Physarum polycephalum secreted chitinase and β-N-acetylhexosaminidase. Chitin added to the medium was depolymerized to N-acetylglucosamine, but this was not used for growth. Spent culture medium contained three enzymes with activity towards 4-methylumbelliferyl-β-N-acetylglucosaminide and these were separated by chromatography on DEAE-cellulose. In previous work β-N-acetylhexosaminidases X and Y were identified. The latter form has now been resolved to yield a new form Z. The Y form can be readily distinguished from both X and Z by its lack of activity towards N,N′-diacetylchitobiose. Comparison of β-N-acetylhexosaminidases X and Z revealed differences in thermal stability, pH optima and K m values. 3,4-Dinitrophenyl-tetra-N-acetylchitotetraoside is hydrolysed by β-N-acetylhexosaminidase Z, but not by the X form of the enzyme.