Karst aquifers with high primary-porosity matrix, such as the Floridan aquifer, have the potential for movement of water between conduits and matrix, with important implications for karst development and the maintenance of groundwater quality. The Santa Fe River Sink and River Rise conduit system, along with the surrounding unconfined Floridan aquifer in north-central Florida, provides a study area to test and quantify conceptual models of exchange between conduits and matrix. The Santa Fe River sinks underground and flows for ∼5 km before reemerging at a first-magnitude spring, the River Rise. During February and March 2003, we recorded discharge rates into the Santa Fe River Sink and out of the River Rise along with hydraulic heads at the River Sink, River Rise, and matrix monitoring wells. Comparison of conduit and monitoring-well hydraulic heads allowed us to track the changes in hydraulic gradient between conduits and wells as a discharge peak passed through the conduits, and the observed head differences between the wells and conduit show a linear relationship with gains and losses of water from the conduit system. The responses of heads at three of the monitoring wells to changes in head within the conduits suggest a transmissivity between 950 and 160,000 m²/d, and analysis suggests that the values depend on the scale of measurement. These results demonstrate the potential for transmissivity determinations in karst aquifers by passive monitoring and are consistent with previous observations that transmissivity of karst aquifers varies with the scale over which it is measured.