When Phanerochaete chrysosporium was grown with laminarin (a beta-1,3/1,6-glucan) as the sole carbon source, a beta-1,3-glucanase with a molecular mass of 36 kDa was produced as a major extracellular protein. The cDNA encoding this enzyme was cloned, and the deduced amino acid sequence revealed that this enzyme belongs to glycoside hydrolase family 16; it was named Lam16A. Recombinant Lam16A, expressed in the methylotrophic yeast Pichia pastoris, randomly hydrolyzes linear beta-1,3-glucan, branched beta-1,3/1,6-glucan, and beta-1,3-1,4-glucan, suggesting that the enzyme is a typical endo-1,3(4)-beta-glucanase (EC 3.2.1.6) with broad substrate specificity for beta-1,3-glucans. When laminarin and lichenan were used as substrates, Lam16A produced 6-O-glucosyl-laminaritriose (beta-D-Glcp-(1->6)-beta-D-Glcp-(1->3)-beta-D-Glcp-(1->3)-D-Glc) and 4-O-glucosyl-laminaribiose (beta-D-Glcp-(1->4)-beta-D-Glcp-(1->3)-D-Glc), respectively, as one of the major products. These results suggested that the enzyme strictly recognizes beta-D-Glcp-(1->3)-D-Glcp at subsites -2 and -1, whereas it permits 6-O-glucosyl substitution at subsite +1 and a beta-1,4-glucosidic linkage at the catalytic site. Consequently, Lam16A generates non-branched oligosaccharide from branched beta-1,3/1,6-glucan and, thus, may contribute to the effective degradation of such molecules in combination with other extracellular beta-1,3-glucanases.