Phospholipase C (phosphatidylcholine phosphohydrolase, EC 3.1.4.3) and lipase (EC 3.1.1.3) activities were detected in the supernatant fluid of Pseudomonas fluorescens strain D cultures. A combination of ultrafiltration and successive chromatography through columns of Sephadex G-75 and DEAE-cellulose was used to purify the phospholipase C over 700-fold from the culture medium, with 28.5% yield. The purified enzyme appeared as a single band after polyacrylamide gel electrophoresis. The apparent molecular mass of the phospholipase C was 36,000 daltons when estimated by gel permeation chromatography. The purified enzyme hydrolysed phosphatidylcholine more efficiently than phosphatidylethanolamine. The synthetic substrate p-nitrophenylphosphorylcholine, phosphatidylinositol or sphingomyelin were not hydrolysed. Hydrolysis of phosphatidylcholine was inhibited by EDTA (1mM) and stimulated by Zn2+, Mg2+ ions and detergents. These properties of the enzyme indicate that it is distinct from the previously reported Ps. fluorescens phospholipase C.