Cell-free extracts, membranous fractions, and cell wall preparations from Schizosaccharomyces pombe were examined for the presence of (1 --> 3)-beta-, (1 --> 3)-alpha-, and (1 --> 6)-beta-glucanase activities. The various glucanases were assayed in cells at different growth stages. Only (1 --> 3)-beta-glucanase activity was found, and this was associated with the cell wall fraction. Chromatographic fractionation of the crude enzyme revealed two endo-(1 --> 3)-beta-glucanases, designated as glucanase I and glucanase II. Glucanase I consisted of two subunits of molecular weights 78,500 and 82,000, and glucanase II was a single polypeptide of 75,000. Although both enzymes had similar substrate specificities and similar hydrolytic action on laminarin, glucanase II had much higher hydrolytic activity on isolated cell walls of S. pombe. On the basis of differential lytic activity on cell walls, glucanase II was shown to be present in conjugating cells and highest in sporulating cells. Glucanase II appeared to be specifically involved in conjugation and sporulation since vegetative cells and nonconjugating and nonsporulating cells did not contain this enzyme. The appearance of glucanase II in conjugating cells may be due to de novo enzyme synthesis since no activation could be demonstrated by combining extracts from vegetative and conjugating cells. Increased glucanase activity occurred when walls from conjugating cells were combined with walls from sporulating cells. Studies with trypsin and proteolytic inhibitors suggest that glucanase II exists as a zymogen in conjugating cells. A temperature-sensitive mutant of S. pombe was isolated which lysed at 37 degrees C. Glucanase activity was higher in vegetative cells held at 37 degrees C than cells held at 25 degrees C. Unlike the wild-type strain, this mutant contained glucanase II activity during vegetative growth and may be a regulatory mutant.