Two different enzymes which cause the breakdown of nutritional chitin in the hunting spider Cupiennius salei were purified to homogeneity and characterized. One chitinase (EC 22.214.171.124) was enriched about 80-fold (molecular weight 48 000). Its action upon solubilized chitin yields N,N′- diacetylchitobiose (88%), trimers (5%) and monomers (7%) of N- acetylglucosamine , respectively. The enzyme has a pH-optimum around pH 7.2 and it liberates about 30 nmol of N,N′- diacetylchitobiose from chitin per mg protein per s. The enthalpy of activation is 9.5 kcal/mol at 30°C. N,N′- Diacylchitobiose , in turn, is cleaved by a specific β-N- acetylglycosaminidase (EC 126.96.36.199), which hydrolyzes N,N′- diacetylchitobiose , the 4-methylumbelliferyl β-derivatives of N- acetylglucosamine and N- acetylgalactosamine , and p- nitrophenyl-β-N- acetylglucosaminide at nearly equal rates (approx. 250 nkat/mg protein). The pH optima range around pH 5.4 for the chromogenic substrates and around pH 6.5 for N,N′- diacetylchitobiose . Heat stability for the enzyme is better at pH 6.5 than at pH 5.4; activity is not inhibited by any of the substrates used. β-N- acetylglucosaminidase was purified about 160-fold (molecular weight 108 000) and is characterized by kinetic and thermodynamic parameters and inhibitors. Through inhibition studies with N- acetylglucosamine-1 → 5 lactone, it could be shown that all activities pertain to only one enzyme and bind to an identical active centre.