Phospholipase C (phosphatidylcholine cholinephosphohydrolase, EC 3.1.4.3.) was secreted in the culture supernatant by Pseudomonas fluorescens strain B. The maximum enzyme release was observed at the early stationary phase of culture growth. The most suitable medium for enzyme production was found to be casein soy broth containing additional 0.5% xylose and 1mM MgCh2. Phospholipase C from Pseudomonas fluorescens was purified 37 fold with 42% yield by a combination of ultrafiltration and Sephadex G75 column chromatography.