The transphosphatidylation and hydrolytic activities of phospholipase D in culture supernatants of soil isolates were evaluated by a specific spectrophotometric method for quantitative determination using an artifical substrate, phosphatidyl-p-nitrophenol. Phospholipase D from strain TH-2 showed the highest specific activity and ratio of transphosphatidylation activity to hydrolytic activity among those from the eight soil isolates and commercial Actinomycetes phospholipase D.