THE natural fluorescence properties of sea water provide a means of elucidating the complex chemical composition and diverse sources of dissolved organic matter (DOM) in sea water1–6. The positions of excitation and emission maxima for a wide range of natural water samples show remarkable similarity7. High-sensitivity fluorescence spectroscopic studies8 have shown recently that emission maxima for marine and coastal waters differ by 20 nm when the excitation wavelength is 313 nm. Here we present evidence from three-dimensional excitation emission matrix (EEM) spectroscopy that at least three fluorophores are present in waters of the Black Sea. Distinct changes in the relative abundance of these fluorophores are observed as a function of depth. We suggest that three-dimensional fluorescence spectroscopy can be used to distinguish between different types and sources of DOM in natural waters. These findings may have important applications in the field of remote sensing of phytoplankton pigments. For example, a better understanding of the sources of DOM components will help in correcting9,10 remotely sensed data for the presence of gelbstoff (yellow-coloured DOM11, which plays an important part in radiation absorption by surface waters).