Abstract The activity and the stability of the β-galactosidases from Thermus sp strain T2 and Kluyveromyces fragilis have been compared. Both enzymes have been partially purified by gel permeation chromatography, determining their molecular weights too. The influence of several metal cations and some buffers on the activity of the enzymes has been tested. The specificity of the enzymes for galactosyl moieties and β-bonds has been established by testing their activity on several synthetic chromogenic substrates and disaccharides. Also, it has been determined that both enzymes showed a remarkable hydrolytic activity and a weak transgalactosilation activity, even in the presence of high concentrations of lactose. The stability of both enzymes in soft and extreme conditions of pH and temperature and in the presence of aggressive chemicals (organic miscible solvents, oxygen peroxide, surfactants and urea) was studied. The thermophilic enzyme showed a higher resistance to hydrophobic agents and a higher stability at different temperatures, pHs and chemical conditions. However, the enzyme of Thermus was less stable in the presence of oxygen peroxide, showing that some residues important for its stability were affected by oxidation. Kinetic studies on the ONPG hydrolysis with both enzymes were carried out in a wide range of temperatures and substrate and product concentrations. The data obtained at all the temperatures were fitted by a nonlinear technique to different kinetic models and two of them were selected to describe the reaction catalysed by the enzymes. The enzyme from K. fragilis was strongly inhibited by o-nitrophenol in a acompetitive way but it was weakly and competitively inhibited by galactose. The thermophilic enzyme was competitively inhibited by galactose much strongly than its mesophilic counterpart but the inhibition did not change with the temperature.