The substrate specificities of three molluscan sulfatases (E.C. 220.127.116.11; snail, abalone, and limpet origins) were investigated with assorted p-nitrophenyl (pNP) di-O-sulfonated β-D-galactopyranosides and β-lactosides [3,6-SO3Gal (1), 3′,6′-SO3Lac (2), 4, 6SO3Gal (3), 2,6-SO3Gal (4), 3,4-SO3Gal (5), and 3,6-SO3GalNAc (6); Ac, acetyl; Gal, galactose; Lac, lactose] together with mono-O-sulfonated β-D-galactopyranoside [pNP 3SO3-Gal (7)] and tri-O-sulfonated α-D-galactopyranoside [2,3,6-SO3-α-Gal (11)]. Some notable differences between the substrate specificity of the three sulfatases were disclosed; snail sulfatase hydrolyzed the 3O- and 2O-sulfo groups of 1 and 4, respectively, to afford 6SO3Gal (9) in high yields, while the abalone enzyme did not act on 4. Only the limpet enzyme could cleave the 3O-sulfo groups of 7 to give pNP β-galactoside. In contrast, every enzyme could utilize 11 as a good substrate to afford a mixture of 6SO3-α-Gal (13) and 2,6-SO3α-Gal (12). None of the enzymes could cleave the O-sulfo groups of 5 and 6, which indicates that a primary 6O-sulfo group tends to promote the enzymatic hydrolysis of O-sulfo groups at the secondary positions.