Abstract Estimation method of phospholipase C in soil is described. This method involves colorimetric estimation of p-nitrophenol released by phospholipase C activity when soil is incubated in Tris-maleate buffer with p-nitrophenylphosphorylcholine (PNPC) and toluene at 30°C for 2 hr. Autoclaving completely destroyed PNPC-hydrolytic activities of several cultivated soils. Optimum pH of the enzyme activity was about 9. The K m value for PNPC was 3.9- 5.6 × 10-3 M. p-Nitrophenol released from PNPC by phospholipase D and phosphomonoesterase during incubation within 3.5 hr is negligible at least in the case of uSing the method described. The activity of phospholipase C in soil was decreased by air-drying in various ranges. The results from antibiotic treatment of soil suggested that bacteria were the important producer of phospholipase C in soil and that in some soil the enzyme of fungal origin might partly constitute the activity of soil phospholipase C.