Technical aspects of the separation of aqueous two-phase systems in a commercial separator were studied in detail. For the Gyrotester B, the smallest available separator, a flow rate of 200 ml/min and a length of the regulating screw in the outlet port of 13.5 mm were found as optimal operation parameters for the separation of a poly(ethylene glycol) (PEG)/dextran two-phase system. In the presence of cells and cell debris the characteristics of the carrier two-phase systems are changed, most notably the phase ratio. Nevertheless good separation and high throughput can be maintained up to 30% wet cell material in the complete system. Using this method the enzyme pullulanase was extracted from 6.65 kg Klebsiella pneumoniae in 88% yield in a single step in less than 2 hr. A yield of 90% was predicted for this step based upon laboratory data, indicating that the performance of the extraction and separation can be calculated with the necessary accuracy and the further scale-up of the process should be accomplished quite easily. The hydrophilic polymers constituting the phase system will often stabilize the enzymes, so that the separation can be carried out at room temperature without extensive cooling. The method of enzyme solubilization or cell disruption is not decisive for the successful extraction of the enzymes, the only limitation being the necessity to find a suitable two-phase system where the desired product and the cells or cell debris will partition in opposite phases. This is shown for a-glucosidase from Saccharomyces carlsbergensis and three aminoacyl-tRNA-synthetases from Escherichia coli. The results obtained demonstrate that aqueous two-phase systems can be separated in commercially available separators with high capacity and efficiency. It can be expected that the advanced separation technology available from chemical engineering studies can also be used for the development of large-scale isolation processes for enzymes involving liquid-liquid partitions.