Six strains of Pediococcus pentosaceus and two of P. acidilactici had intracellular/3-galactosidase (fl-gal) activity when grown in the presence of lactose; all but two strains of P. pentosaceus and one of P. acidilactici had such activity when grown in the presence of glucose. Synthesis of/3-gal by P. pentosaceus ATCC 25 745 was inducible with lactose, galactose, melibiose, lactobionic acid and possibly cellobiose but not with glucose, sucrose, maltose, glycerol, fructose or mannose. Lactose, galactose and possibly maltose, melibiose and lactobionic acid but not glucose, sucrose, glycerol, cellobiose, fructose or mannose induced/3-gal synthesis by P. acidilactici ATCC 25740. Synthesis of /3-gal was partially inhibited in P. pentosaceus ATCC 25745 and P. aeidilactici ATCC 25 740 by glucose added to the medium during growth in the presence of galactose or lactose. Isopropyl fl-D-thiogalactopyranoside failed to induce synthesis of/3-gal by either strain during growth on glucose./3-Gal from P. pentosaceus ATCC 25 745 had a molecular weight of 66,000 and activity optima of pH 6.5 and 45 ° C. Activity of the enzyme was stimulated by reducing agents, Mg 2+, Mn 2+, Zn 2+ and Co 2+ but not by Ca 2+, and was markedly inhibited by ethylenediaminetetraacetate (EDTA), HgCl2, 1,10-phenanthroline, and an oxidizing agent. The K,~ values of the enzyme for o-nitrophenyl-/3-D-galactopyranoside and lactose were 3.07 and 7.0 mM, respectively, suggesting its low affinity for lactose.