Phospholipase D from Dacus carota (carrot) was purified by subjecting it to three-phase partitioning. The single step of three phase partitioning led to 13-fold purification with an activity recovery of 72%. SDS-PAGE analysis showed a single band with minimum molecular weight corresponding to nearly 60 kDa. The purified enzyme had a pH optimum in the range of 6.0-6.5 and was unstable above 30 degreesC. Kinetic studies showed a K-m value of 9.5 mM and a V-max of 0.35 mL min-l. The enzyme purified by three phase partitioning was found to resolve into two isoenzymes on a DEAE cellulose column.