Thermoacidophiles are microorganisms capable of optimum growth under a combination of high temperature and low pH. These microorganisms are a rich source of thermo- and acid- active/stable glycosyl hydrolases. Such enzymes could find use as novel biocatalysts in industrial processes, as operation at elevated temperature can increase substrate solubility, decrease viscosity, and reduce the risk of microbial contamination. We report the purification and characterization of an intracellular beta-galactosidase from the thermoacidophile Alicyclobacillus vulcanalis DSM 16176. The enzyme was purified 110-fold, with a 5.89% yield. Denatured (83.7 kDa) and native (179 kDa) molecular masses were determined by SDS-PAGE and gel filtration, respectively, and suggest the enzyme functions as a homodimer. LC-MS/MS analysis confirmed identity, and bioinformatic analysis indicates the enzyme to be a member of the glycosyl hydrolase family 42 (GH42). Highest activity was measured at 70 degrees C and pH 6.0. The K-m on the substrates ONPG and lactose were 5 and 258 mM, respectively. This enzyme is thermostable, retaining 76, 50, and 42% relative activity after 30, 60, and 120 min, respectively, at 70 degrees C. This property could lend its use to high-temperature industrial processes requiring a thermo-active beta-galactosidase.