Abstract Phospholipid fatty acid (PLFA) analysis has become a popular and powerful method for characterizing soil microbial communities. However, little work has been done to compare different extraction procedures. In this study, the effects of single or consecutive extractions, varying soil weights, alternative solid phase extraction (SPE) tubes and a comparison of extractions from fresh soil and that from soil previously stored freeze-dried at − 70 °C for 1 year were determined. About 90–95% of PLFAs were extracted by the method used in this paper. More individual PLFAs and changes in PLFA peak distributions (profiles) were found with increasing soil sample weights, which suggests that for each set of experiments, the soil weight should be constant. Home-made SPE tubes used in this study can give almost as good results as the commercial standard ones, which significantly decreased costs. The total PLFA concentrations decreased by more than 28% following storage of the freeze-dried soils for 1 year at − 70 °C. The PLFA profiles also changed during storage over this period, suggesting that PLFA analyses are best done as soon as possible after sampling.