Karst and fractured rock aquifers are primary sources of drinking water in the Appalachian region, even though most are inherently susceptible to surface-derived contamination. Many of the obstacles to water supply protection in such systems could be alleviated through the use of tracer testing to delineate recharge areas and surface-to-groundwater connections. Tracer testing is currently under-utilized, however, due to public safety concerns and ambivalence on the part of regulatory agencies. This study aimed to address this issue through a characterization of the fluorescent properties of typical carbonate aquifers for the purpose of refining the timing and design of lowconcentration dye tracer tests. The subjects of this study were community water supply wells and springs in the Valley and Ridge Province of East Tennessee. Although each source exhibited a different degree of contamination potential, background fluorescence could primarily be attributed to natural dissolved organic matter (DOM) rather than manmade fluorophores. Seasonal data were collected for 23 groundwater sources during a multi-year drought (2006-2007). Untreated water samples were analyzed via fluorescence spectroscopy to identify spatial and temporal variations in the fluorescent properties of high-risk and lowrisk water supply sources. Fluorescence exhibited seasonal patterns and fluctuations in DOM quality relative to rain events. Fulvic acid-like spectra were detected most consistently in all sources and exhibited maximum fluorescent intensity in the winter months. Humic acid-type fluorescence appeared in springs and high-risk wells only after major rainfall. For all sources, DOM spectra gradually shifted towards lower molecular weight fluorophores, which was interpreted as a reflection of diminishing humification and increasing generation of proteins with warmer weather and drier conditions. Variations in fluorophore composition and concentration were generally more pronounced in springs and moderate- to high-risk wells than in low-risk wells, although all sources exhibited some unique characteristics. The results indicate that extended background fluorescence characterization could enhance the design of tracer tests and sampling strategies for waterborne contaminants such as cyanotoxins, VOCs, and microbial pathogens. This study also highlights the potential benefits of monitoring DOM fluorescence as a natural tracer for source water and wellhead protection.