We screened culture filtrates of various microorganisms for hydrolytic enzyme inhibitors and report the characterization of pyridindolol, an inhibitor of β-galactosidase in acidic conditions. A strain MD401-C5, isolated from soil in Minamisaku, Nagano Prefecture and classified as Streptomyces alborerticillatus, was used for production. Pyridindolol was produced via shaking or tank fermentation and purified by active carbon adsorption, elution with 0.05 N HCl in 50% acetone, ethyl acetate extraction, Sephadex LH-20 column chromatography, and recrystallization from methanol-ethyl acetate. The purified compound is colorless needles with a melting point of 167~168°C, [α]25n -49' (c 1.0, MeOH), molecular formula C14H14O3N2 (molecular weight 258.27), and solubility in polar solvents (e.g., methanol, dimethylsulfoxide) but not in non-polar solvents (e.g., benzene, chloroform). It inhibits bovine liver β-galactosidase at pH 4.2 with an ID50 of 2.0 μg/ml, but shows no inhibition against sialidase (from Clostridium perfringens, Streptomyces, or influenza virus) or β-galactosidase at pH 7.0 at concentrations >250 μg/ml. Pyridindolol (100 μg/ml) exhibits no antibacterial or antifungal activity and has low toxicity—intraperitoneal injection of 500 mg/kg in mice caused no death. Its structure was determined as 1-[1(R),2-dihydroxyethyl]-3-hydroxymethyl-9H-pyrido[3,4-b]indole.