The aim of the present research was to investigate the influence of culture conditions on the levels of β-galactosidase (EC 3.2.1.23) activity produced by Kluyveromyces strains. Interest was focused on evaluating enzyme activity levels when lactose or cheese whey was employed as substrate in culture medium formulation. From an overall look at the obtained results, the tested strains were found to be able to produce β-galactosidase at promising levels. The use of cheese whey, either for strain maintenance and production trials, allowed to obtain a high cell yield associated with β-galactosidase production. The maximum β-galactosidase volumetric activity, EA max 66.5 IU/ml, corresponding to 3184 EA spec IU/g cell dw, was obtained with K. marxianus MIM 782 at 37 °C and 72 h incubation.