A 60 kDa phospholipase D (PLD) was obtained from Streptomyces olivochromogenes by one-step chromatography on Sepharose CL-6B. Maximal activity was at pH 8 and 75 degrees C and the enzyme was stable from pH 7 to 13 and from 55 to 75 degrees C. Thermal and pH stability with temperature optimum of the enzyme were highest among Streptomyces PLDs reported so far. The activity was Ca(2+)-dependent and enhanced by detergents. The Km and Vmax values for phosphatidylcholine were 0.6 mM and 650 mumol min(-1) mg(-1), respectively. In addition, the enzyme also revealed transphosphatidylation activity, which was optimum at pH 8 and 50 degrees C. The first 15 amino acid residues of the N terminal sequence were ADYTPGAPGIGDPYY, which are significantly different from the other known PLDs. The enzyme may therefore be a novel PLD with potential application in the lipid industry.