Minipiezometers installed at different vertical levels within the streambed (20–140 cm) were used to study temporal and spatial variation in the dissolved organic carbon (DOC) content of streamwater and groundwater in three southern Ontario streams. Groundwater, as represented by our streambed samples, contained considerable quantities of DOC but variation between replicate samples was high. Diel fluctuations in DOC content of streamwater were consistent with daytime autochthonous production and night-time uptake by heterotrophs. Water from the streambed neither consistently diluted nor enhanced streamwater levels of DOC. At some stations, DOC variation with depth, including streamwater, seemed to be largely random. At other stations, DOC concentrations from the deepest piezometers were consistently higher than concentrations at intermediate depths, suggesting a loss of DOC from deeper waters to overlying sediments. However, at these stations DOC concentrations were highest at 20 cm and at the surface. Interflow delivery of DOC to the shallow layers of the streambed may be a significant source of carbon for a stream ecosystem, especially in agricultural areas. Late summer diel fluctuations at one station may be related to changing patterns of intermixing of stream and groundwater in the upper layers of the streambed as governed by velocity heads, convective currents and evapotranspiration.