Mannases have industrial uses in food and pulp industries, and their regulation may influence development of the mushrooms of commercially important basidiomycetes. We expressed an Agaricus bisporus cel4 cDNA, which encodes a mannanase, in Saccharomyces cerevisiae and Pichia pastoris. CEL4 had no detectable activity on cellulose or xylan. This gene is the first isolated from this economically important fungus to encode a mannanase. P. pastoris secreted about three times more CEL4 than S. cerevisiae. The removal of the cellulosebinding domain of CEL4 lowered the secreted specific activity by P. pastoris by approximately 97%. The genomic sequence of cel4 was isolated by screening a cosmid library of A. bisporus C54-carb8. The open reading frame was interrupted by 12 introns. The level of extracellular CEL4 increases dramatically at the postharvest stage in compost extracts of A. bisporus fruiting cultures. In laboratory liquid cultures of A. bisporus, the activity of CEL4 detected in the culture filtrate reached a maximum after 21 days. The levels of CEL4 broadly mirrored the levels of enzyme activity. In the Solka floc-bound mycelium, CEL4 protein showed a maximum after 2 to 3 weeks of culture and then declined. Changes in CEL4 activity during fruiting-body development suggest that hemicellulose utilization plays an important role in sporophore formation. The availability of the cloned gene will further studies of compost decomposition and the extracellular enzymes that fungi deploy in this process.␤-Mannanase enzymes (endo-1,4-␤-D-mannanase, mannan endo-1,4-␤-mannosidase; EC 3.2.1.78) are endohydrolases that catalyze the random hydrolysis of ␤-1,4-mannosidic linkages in the main chain of galactomannan, glucomannan, galactoglucomannan, and mannan (20). These enzymes are of particular interest in ligninolytic fungi, where there is a complex interplay between the processes of degradation of lignin, cellulose, and hemicellulose (including the mannans). There are possible biotechnological applications. ␤-Mannanase can be used to bleach pulp, to reduce the viscosity of instant coffee, and for the clarification of fruit juices and wines (35). At the same time, the role of hemicellulases in wood decay and the breakdown of leaf litter remains poorly understood. The cultivated mushroom Agaricus bisporus, a ligninolytic leaf litter degrader (8), is the source of the mannanase described in this paper.The deduced CEL4 amino acid sequence shows that the encoded protein has a modular structure (40). There is a signal peptide at the N terminus, typical of proteins that are secreted into the medium, a catalytic domain, and a linker rich in serine, proline, and threonine that separates a cellulose-binding domain from the catalytic domain. The catalytic domain of CEL4 had the most amino acid sequence similarity with ascomycete mannanases from Aspergillus aculeatus (5) and Trichoderma reesei (28), which belong to glycosyl hydrolase family 5 (43 and 42%, respectively). Therefore, based on amino acid similarities of the catalytic dom...