An investigation was conducted into the use of phospholipid fatty acid (PLFA) profiles to follow temporal shifts in microbial community structure. A laminar pot was designed to allow the harvest of an intact layer of rhizosphere soil 0-2<space>mm from plant roots without the removal of the root material itself. Agrostis stolonifera plants were grown under controlled conditions in environmental chambers for 70, 90, 120 and 160 days. A subset of pots was fertilised with N/P/K after 100 days. Over the 90 days between the first and last harvests microbial biomass declined by 20%. At 70 days respiration was approximately 50% higher in the rhizosphere compared to the bulk soil. This difference had declined to <10% by the end of the experiment. PLFA analysis showed significant changes in microbial community structure with time. In particular, the unsaturated fatty acids commonly found in Gram-negative bacteria and fungi increased with age. Principal component analysis (PCA) of fatty acid data showed: separation of bulk and rhizosphere soils from 90 days onwards, and; fertiliser addition and rhizosphere development produced similar shifts in community structure.