For simplification of cell disruption and the subsequent aqueous two-phase separation process, simultaneous release and purification of beta-galactosidase from a recombinant Escherichia coli were investigated using a PEG-phosphate aqueous two-phase system containing glycine. The enzyme was released in the two-phase system by the addition of glycine, the percentage of the release increasing with incubation time. The effects of PEG and phosphate concentrations on the release were investigated in a buffer solution. Although the release was enhanced by the addition of PEG, phosphate repressed it. In order to ascertain the optimum operational conditions, the effects of various parameters on the partition coefficient (top/bottom) of the enzyme were investigated. The enzyme was preferentially partitioned to the top phase in a 13% PEG 6000-10% phosphate system, but the partition coefficient was significantly decreased by the addition of NaCl. It was slightly decreased by adding the supernatant of a sonicate of the host cells, but significantly increased with decreasing PEG molecular weight. The addition of glycine to the two-phase system favorably enhanced the partition of the enzyme to the top phase.