The usefulness of measuring neutral lipid fatty acids (NLFAs) and phospholipid fatty acids (PLFAs) separately in order to interpret perturbation effects on soil and compost microorganisms has been studied. Initially the NLFA/PLFA ratios were studied in different soils. Low ratios were found for fatty acids common in bacteria, especially for cyclopropane fatty acids. Higher ratios were found for fatty acids common in eukaryotic organisms such as fungi (18:1omega9 and 18:2omega6,9) or in saturated fatty acids, common to many types of organisms. Adding glucose to a forest soil increased the amounts of the fungal NLFAs 18:1omega9 and 18:2 omega6,9 up to 60 and 10 times, respectively, after 10 days, followed by a gradual decrease. After 3 months incubation, higher levels of these NLFAs were still found compared with the control samples. Adding glucose together with nitrogen (N) and phosphorus (P) resulted in no increase in NLFAs but a 10-fold increase in the PLFAs 18:1omega9 and 18:2omega6,9. Thus, the NLFA/PLFA ratios for these fatty acids were lower than in the no-addition control when glucose was added together with N and P, but higher when glucose was added alone, even 3 months after the addition. Adding N+P without glucose did not affect the NLFA/PLFA ratio for any fatty acid. Increasing NLFA/PLFA ratios for the fungal fatty acids were also found with time after the thermophilic phase in a compost, indicating increased availability of easily available carbon.