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Interstitial water, faunal samples, and hydrogeological data were collected beneath a riffle on the Speed River, southern Ontario, Canada. The purpose was to identify the location and seasonal fluctuation of the hyporheic/groundwater interface and to examine several aspects of water mass chemical signatures and the dynamics of the interstitial fauna. Concentration discontinuities in several water chemistry parameters delineated the chemical boundary between the true groundwater and hyporheic habitats. The groundwater mass was characterized as having higher levels of ammonium, alkalinity, and conductivity, and lower nitrate levels. Differences in water chemistry between the hyporheic and groundwater zones persisted throughout the year, though no single variable differed quantitatively between these two zones on all occasions. The location of the chemical discontinuity varied seasonally. Whereas hyporheic and groundwater faunal subunits of the interstitial community were identified and the location of the subunits coincided with the chemical breaklines, response to shifts in the position of the hyporheic/groundwater interface was taxon rather than subunit based. Fauna therefore provided poor spatial resolution in terms of pinpointing the location of the interface. Boundary fluctuation coincided with extremes in seasonal discharge patterns and was regulated by the relative strength of the upward force of baseflow and the downward force of advecting surface water. Identifying patterns of fluctuation of the hyporheic/groundwater interface, and consequently hyporheic habitat volume, may have important consequences for the storage, retention, and cycling of nutrients in lotic ecosystems.