Endospores are heat-resistant bacterial resting stages that can remain viable for long periods of time and may thus accumulate in sediments as a function of sediment age. The number of spores in sediments has only rarely been quantified, because of methodological problems, and consequently little is known about the quantitative contribution of endospores to the total number of prokaryotic cells. We here report on a protocol to determine the number of endospores in sediments and cultures. The method is based on the fluorimetric determination of dipicolinic acid (DPA), a spore core-specific compound, after reaction with terbium chloride. The concentration of DPA in natural samples is converted into endospore numbers using endospore-forming pure cultures as standards. Quenching of the fluorescence by sediment constituents and background fluorescence due to humic substances hampered direct determination of DPA in sediments. To overcome those interferences, DPA was extracted using ethyl acetate prior to fluorimetric measurements of DPA concentrations. The first results indicated that endospore numbers obtained with this method are orders of magnitude higher than numbers obtained by cultivation after pasteurization. In one of the explored sediment cores, endospores accounted for 3% of all stainable prokaryotic cells.