The excretion of extracellular polymeric substances (EPS) by an axenic culture of the benthic diatom Cylindrotheca closterium was investigated. Two sequential extraction steps proved to be sufficient to remove the bulk of the EPS present. Soluble EPS was recovered by a simple centrifugation step and represented a fraction that was not or was only loosely associated with diatom cells. For the extraction of bound EPS, different procedures were compared. The best results were obtained using distilled water as extraction solvent (1 h, 30 °C). The sugars that were recovered using this procedure were typically associated with aggregates of diatoms. In addition to the distinct differences in localization of the different types of EPS, their temporal dynamics differed in relation to the light–dark cycle. Soluble EPS were continuously released into the medium at a rate of 1·6 pg cell−1 day−1. In contrast, the production of bound EPS was highly light-dependent. In the dark, this bound EPS rapidly disappeared, probably as the result of its utilization by the diatoms.