Specific fatty acids from phosphatidylglycerol (PG) and phosphatidylethanolamine (PE) recovered from a per 13C-labeled bacteria can be detected in environmental samples and used as measures of bacterial transport in the subsurface. Detection of palmitic acid (16:0) and oleic acid (18:1) at m/z 271 (255+16) and 299 (281+18) as negative ions in PG and PE separated by high performance liquid chromatography (HPLC) and detected after up-front collisionally induced dissociation (CID) utilizing electrospray (ES) mass spectrometry (MS) provided sufficient sensitivity and specificity for detection in the presence of the indigenous microbiota. Application of tandem mass spectrometry (MS/MS) in the multiple reaction monitoring (MRM) was use to monitor selected transitions. MRM can increase the sensitivity so that polar lipids recovered from cell densities currently at about 104 cells/sample can be detected. This technology provides a non-intrusive mechanism for monitoring the distribution of bacteria added to accelerate in situ bioremediation of subsurface sediments. © 2001 Elsevier Science B.V.