beta-Galactosidase activity of CTAB-pemmeabilized K. fragilis depends upon the cell to detergent ratio and the pH of the permeabilizing medium. Maximum beta-galactosidase activity was observed when the cell to detergent ratio was 1:0.01 at neutral pH. A simple and single step method for the preparation of whole cell beta-galactosidase is described which involves adjusting the pH of the growth medium at the beginning of the stationary phase to 7.0, addition of CTAB directly to the growth medium at 1% concentration of biomass (wet weight), before harvesting cells by centrifugation. This whole cell enzyme activity readily hydrolysed high concentrations of lactose in buffer and whey.