Phospholipid fatty acid analysis is widely used to characterize soil microbial communities.  The procedure is, however, lengthy, expensive, and uses large volumes of organic solvents. We have developed a streamlined protocol to reduce costs and simplify processing large numbers of samples. Lyophilized soil samples (1-2 g) are extracted with a modified Bligh-Dyer solvent system in test tubes. Phases are separated in test tubes and the organic phase evaporated under vacuum. Lipid classes are separated by chromatography on a 96-well solid phase extraction plate, with column eluates collected in glass vials in a 96-well format,  and the phospholipids are dried under vacuum. All further processing steps, including transesterification, extraction of the resulting fatty acid methyl esters, drying, and resuspension, are carried out in glass vials in a 96-well format, using multichannel pipettors for liquid transfers. The final solution of fatty acid methyl esters in hexane (50 microliters) is transferred to GC vials with limited volume inserts and analyzed by GC using the Sherlock system (MIDI Inc., Newark, DE, USA). Samples are injected at a split ratio of 30:1 and fatty acids from 10 to 24 carbons long are identified against a naming table which currently contains 114 entries. 96 previously lyophilized samples can be prepared in two days. With a GC run-to-run cycle time of 14 minutes, 96 samples plus calibration and re-calibration runs can be analyzed in 25 hours. This protocol should substantially reduce labor and material costs and expedite the use of PLFA to analyze large numbers of samples.