Rationale Compound‐specific isotope analysis (CSIA) of nitrogen in amino acids has proven a valuable tool in many fields (e.g. ecology). Several intact polar lipids (IPLs) also contain nitrogen, and their nitrogen isotope ratios have the potential to elucidate food‐web interactions or metabolic pathways. Here we have developed novel methodology for the determination of δ 15 N values of nitrogen‐containing headgroups of IPLs using gas chromatography coupled with isotope‐ratio mass spectrometry. Methods Intact polar lipids with nitrogen‐containing headgroups were hydrolyzed and the resulting compounds were derivatized by (1) acetylation with pivaloyl chloride for compounds with amine and hydroxyl groups or (2) esterification using acidified 2‐propanol followed by acetylation with pivaloyl chloride for compounds with both carboxyl and amine groups. The δ 15 N values of the derivatives were subsequently determined using gas chromatography/combustion/isotope‐ratio mass spectrometry. Results Intact polar lipids with ethanolamine and amino acid headgroups, such as phosphatidylethanolamine and phosphatidylserine, were successfully released from the IPLs and derivatized. Using commercially available pure compounds it was established that δ 15 N values of ethanolamine and glycine were not statistically different from the offline‐determined values. Application of the technique to microbial cultures and a microbial mat showed that the method works well for the release and derivatization of the headgroup of phosphatidylethanolamine, a common IPL in bacteria. Conclusions A method to enable CSIA of nitrogen of selected IPLs has been developed. The method is suitable for measuring natural stable nitrogen isotope ratios in microbial lipids, in particular phosphatidylethanolamine, and will be especially useful for tracing the fate of nitrogen in deliberate tracer experiments. Copyright © 2015 John Wiley & Sons, Ltd.