Enterobacter sp. 3TP2A isolated from a petroleum station was found to produce a novel, highly inducible mesophilic intracellular β-galactosidase in the presence of lactose up to 76.5 U mg -1 . The enzyme was purified to 17.3--fold after gel permeation chromatography with a yield of approximately 11 %. The optimum pH and temperature values of the purified enzyme were found to be 8.0-9.0 and 35 °C, respectively. The molecular weight of the enzyme was approx. 60 kDa with a single band by both SDS-PAGE and native-PAGE, and estimated by gel filtration chromatography. The enzyme was inhibited by Zn 2+ and EDTA, while Cu 2+ had strong inhibitory effect even at low concentrations. Activation by Mg 2+ and inhibition by EDTA show that the enzyme is metal--dependent or a metalloenzyme. The enzyme was slightly activated by 2-mercaptoethanol, while slightly inhibited by iodoacetamide. On the other hand, PCMB inhibited the enzymatic activity to a great extent, whereas it was completely inhibited by N-ethylmaleimide. The V max and K m values were calculated as 0.701 μmol min -1 and 0.104 mM, respectively. The results indicated that the β-galactosidase Enterobacter sp. 3TP2A might well be a good candidate for use in biotechnology, particularly in the area of environment and health.