Phospholipid-derived fatty acids (PLFA) are widely used as chemotaxonomic markers in microbial ecology. In this paper we explore the use of PLFA as chemotaxonomic markers for phytoplankton species. The PLFA composition was determined for 23 species relevant to estuarine phytoplankton. The taxonomic groups investigated were Chlorophyceae, Trebouxiophyceae, Prasinophyceae, Bacillariophyceae, Dinophyceae, Cryptophyceae, Rhodophyceae, Pavlovophyceae and Prymnesiophyceae. Most of these taxonomic groups have a characteristic PLFA composition, although a few contain truly unique PLFA biomarkers. The PLFA composition was used to derive species composition and abundance of the phytoplankton in the Scheldt estuary (border region between Belgium and the Netherlands) in April, July and October 2003, using the matrix factorization program CHEMTAX. The results agree well with results from HPLC-derived pigment analysis of the same samples. The advantages of using PLFA instead of pigments, or rather in addition to pigments, are that (1) information on bacteria can be derived in addition to information on photosynthetic taxa, (2) in some cases more or different taxa can be distinguished and (3) the isotopic composition of PLFA can be measured relatively easily, allowing the measurement of group-specific primary production.