Freeze-dried cells of Meiothermus ruber catalyses cleavage of o-nitrophenyl-β-D-galactopiranoside (oNPβ-gal) and conversion of lactose into glucose and galactose. The permeabilization with 2% toluene, 20% ethanol and 20% acetone increased enzymatic activity from 74.87 U/g of lyophilized cells up to 129.44, 114.38 and 90.19 U/g, respectively. Ethanol was an effective permeabilizing agent and its efficiency was dependent on the concentration, the incubation time and incubation temperature. The Km values for the untreated and permeabilized cells were 2.94 mM and 2.26 mM but Vmax values were 122 µmol/min and 193 µmol/min, respectively. The optimum pH for the β-galactosidase activity in the untreated and permeabilized cells were 6.5 and optimum of temperatures 65C. The stability of enzymatic activity in M. ruber cells incubated for 1 h at pH 6.5 was almost unchanged at temperatures below 65C.