The Biscayne aquifer is a highly transmissive karst limestone that serves as the sole source of drinking water to over two million residents in south Florida. The aquifer is characterized by eogenetic karst, where the most transmissive void space can be an interconnected, touching-vug, biogenically influenced porosity of biogenic origin. Public supply wells in the aquifer are in close proximity to lakes established by surface mining. The mining of the limestone has occurred to the same depths as the production wells, which has raised concerns about pathogen and chemical transport from these surface water bodies. Hydraulic and forced gradient tracer tests were conducted to augment geologic and geophysical studies and to develop a hydrogeologic conceptual model of groundwater flow and chemical transport in the Biscayne aquifer. Geologic and geophysical data indicate multiple, areally extensive subhorizontal preferential flow zones of vuggy limestone separated by rock with a matrix pore system. The hydraulic response from an aquifer test suggests that the Biscayne aquifer behaves as a dual-porosity medium; however, the results of the tracer test showed rapid transport similar to other types of karst. The tracer test and concurrent temperature logging revealed that only one of the touching-vug flow zones dominates transport near the production wells. On the basis of the rising limb of the breakthrough curve, the dispersivity is estimated to be less than 3% of the tracer travel distance, which suggests that the fastest flow paths in the formation are likely to yield limited dilution of chemical constituents.