Deep groundwater samples from a deep borehole in the Canadian Shield, Ontario, Canada, have been analyzed by fluorometry, to determine the difference in character of the natural organic matter (NOM) with depth. This work was done to obtain a set of geochemical characteristics of deep groundwaters at the site. The fluorescence signal is a complex signature of excitation and emission of light from fluorescent molecules which are part of all natural waters. Fluorescent components have characteristic excitation/emission components, defined as a humic-like (C1), fulvic-like (C2), and protein-like (C3); these are found in various proportions in natural samples. Changes in relative fluorescence intensities of these components have been used in the past to determine the origin and/or processes of the NOM between sampling locations. In this work, six samples were taken at different depths, from ~108 to 650 m below the surface in the borehole. The fluorescence signals of the samples showed three main patterns: (1) the shallower samples (~108, 139 and 285 m) had a pattern similar to that of surface groundwaters, dominated by components C1 and C2; (2) the samples in deep groundwaters (~620 and 650 m) had a weak overall signal, dominated by component C3; finally (3) the mid-depth sample (~503 m) had a component pattern intermediate between the shallower and deeper zones. This set of data is consistent with other data for the groundwaters from this borehole, such as chlorinity, suggesting that the three sampling intervals represent three different types of groundwaters.