The attack mechanism of a purified exo-1,3-beta glucosidase (1,3-beta-D-glucan glucohydrolase, EC 3.2.1.58) was investigated by using as a substrate a mixture of two structurally characterized periodate-oxidized and reduced unbranched 1,3-beta-D-glucans which differed only at the reducing terminal. The substrates, derivatives of laminarin, were altered only at the terminals due to resistance of the internal (1 leads to 3)-linked glucosyl residues to periodate oxidation. Each glucan has only a single and identical altered non-reducing terminal per molecule. Upon enzymatic hydrolysis, one molar equivalent of glycerol was produced from the altered non-reducing terminal of each substrate molecule attacked. Using glycerol as an indication of the number of chains acted upon, the quantity of D-glucose produced from the internal residues was used to determine the extent to which a chain was initially attacked. The glucose to glycerol ratio during the course of the hydrolysis indicates that the enzyme proceeds by a multiple-attack mechanism where four glucosyl residues are successively removed per encounter from the non-reducing terminal of each substrate molecule.