Abstract: The activity of phospholipase D (PLD) from Streptomyces chromofuscus in two-phase systems consisting of a PLD containing aqueous phase and a phosphatidylcholine (PC) containing organic solvent phase was investigated. Two-phase systems with highly reproducible segmentation were generated in a continuous-flow system using a capillary segmentor. Between 0.01 and 2 U/mL PLD from Streptomyces chromofuscus were detected in a water/hexane two-phase system. The linear detection range was 0.01 to 0.5 U PLD/mL. The apparent KM of Streptomyces chromofuscus PLD in a water/hexane system was 41.43 µM PC, the apparent Vmax was 3.68 µmol min-1 mg-1. The variation of PLD activity in different organic solvents correlated with the solvent's hydrophobicity. If anionic detergents or primary alcohols were added to the two-phase system, concentration dependent activation of the PLD was observed