Investigation of B-galactosidase production by three yeasts and five bacterial species of lactose-fermenting microorganisms during different incubation periods was carried out. K. fragilis and S. salivarius subsp. thermophiius. in shaken cultures. proved to be the most potent and produced the highest B-galactosidase activity after 24 h incubation. Toluene was the most effective soiventin releasing B-galactosidase from the microbial cells. B-Glactosidase release was increased with the solvent concentration till it reached its maximum activity at2.0%(vlv). Higher concentrations were correlated with a great decrease in B-galactosidase activity. Toluene released B-galactosidase in higher yield after 15 min. The higher contact time was correlated with a great decrease in activity. Maximum B-galactosidase activity was extracted at 37°C during suspensions of cells in 0.1-M phosphate buffer at pH 6.8. [3- Galactosidase release was increased with the increase of the exposure time for sonicalion till it reached its maximum after5minutes. The maximum release off}— galactosidase was after 6 cycles of freezing and thawing of the cell pellets and cells in buffer. Higher activity was obtained when the pellets were used.