1. 1.|It has been shown that the β-1,3-glucan hydrolase (β-1,3-glucan glucanohydrolase, EC 3.2.1.39) from Nicotiana glutinosa has an endo-action pattern and is highly specific for linear β-1,3-glucan substrates. These glucans are depolymerized to yield a mixture of β-1,3-oligoglucosides of degree of polymerization 2–7 and a trace of glucose. 2. 2.|Laminaribiose and -triose are not hydrolysed by the enzyme and laminaritetraose and -pentaose are only slowly attacked. The enzyme does not hydrolyse Claviceps β-glucan and the rate of hydrolysis of O-carboxymethylpachyman (CM-pachyman) decreases as the degree of substitution increases. These findings suggest a requirement for at least three unsubstituted β-1,3-glucosyl residues in substrates of this enzyme. The very limited hydrolysis of the β-1,3;1,4-glucans from cereals is probably due to action at the few consecutive β-1,3-linked glucose residues which are present in these substrates. 3. 3.|No transglycosylase activity was detected. 4. 4.|A method for purifying the Rhizopus arrhizus β-1,3-glucan endohydrolase is described and its action on a series of β-glucan substrates is compared with that of the Nicotiana enzyme. 5. 5.|The Nicotiana enzyme was inhibited by Hg 2+ (0.2 mM), Cu 2+ (20 mM), phenylmercurinitrate (2.0 mM), I 2 in KI (2.0 mM), N-bromosuccinimide (5 mM), 2-hydroxy-5-nitrobenzylbromide (10 mM), N-acetylimidazole (23 mM), carbodiimide (100 mM), and the Lespedeza cuneata cellulase inhibitor (0.2 mg/ml).