Stable-isotope labeling of phospholipid-derived fatty acids (PLFAs) is a potentially powerful technique to study group-specific primary production of phytoplankton, as many algal groups possess a specific PLFA composition, and it is relatively simple to measure the isotopic composition of a large number of PLFAs. Experiments with cultured algae showed that differences exist in labeling among PLFAs and between PLFAs and particulate organic carbon (POC), most likely owing to differences in biosynthesis pathways causing some components of the cell with short pathways to be labeled faster. These differences were constant, however, during the first few hours of incubation, and correction factors were used to convert PLFA labeling to total carbon uptake. In algal cultures, growth rates based on 13C-PLFA labeling agreed well with those based on biomass increases in terms of PLFA concentrations and cell numbers. At two contrasting sites in the Scheldt estuary, PLFA synthesis rates were calculated using 2-h incubations. Group-specific primary production was estimated from PLFA synthesis rates and PLFA compositional spectra of samples and algal taxa using the matrix factorization program Chemtax. When accompanied with studies or information on system-relevant phytoplankton, this sensitive method will be applicable to a wide range of pelagic ecosystems and also to benthic systems such as algal mats.