Phospholipid fatty acid (PLFA) analysis has been widely used to study soil microbial community structure. Soil PLFAs are taken to be indicative of living organisms since phospholipids are assumed to rapidly degrade after cell death. However, the turnover rate of phospholipids has never been accurately quantified. For this purpose, a short incubation experiment was conducted by amending paddy soil with extracted phospholipids, using the ¹³C labeling technique and PLFA analysis to quantify phospholipid degradation. Both bacterial (Methylocystis sp. and Escherichia coli) and fungal (Simplicillium subtropicum) phospholipids had high turnover rates. The half-life (t_1/2) for different phospholipids ranged from 14 to 27 h and the average t_1/2 for total phospholipids was about 20 h at 25 °C but nearly double that at 15 °C. However, phospholipids had a similar turnover rate in a soil with lower microbial biomass (84 mg C kg^-1) compared to a soil with higher microbial biomass (305 mg C kg^-1). Assimilation of ¹³C into other phospholipids was very low but followed the same timescale. Overall, this provides for the first time direct evidence for high turnover rates in soil through the analysis of specific ¹³C-labelled PLFAs and confirms the classic hypothesis that intact phospholipids represent living cells, necessary for the validity of the established PLFA methodology.