Reservoir ecosystems may be supported by considerable input of organic matter originating from the catchment. Thus, the current proportion of allochthonous and autochthonous organic matter available to reservoir biota will be affected by changes in the flushing rate. In a medium-sized submontane reservoir, we examined reaction of the δ13C isotopic signature of fine (10–50 μm) and coarse (>50 μm) fractions of the seston, and of benthic invertebrates, to the reservoir flushing rate. The δ13C signatures of samples varied, and spatially δ13C increased significantly down-reservoir in all instances, becoming less similar to the carbon signature of organic matter of terrestrial origin. Two distinct trends in the correlation between δ13C and flushing rate were identified as follows: (1) fine seston tended to become more 13C-depleted and therefore similar to the carbon signature of terrestrial organic matter; and (2) the 13C signature of coarse seston became enriched with increasing flushing, and also tended to become closer to values of terrestrial organic matter. Benthic assemblages did not display any clear association with flushing rates. Such reactions in different components of the food web to allochthonous carbon input and the rate of reservoir flushing may be a common feature of other comparable Central European reservoirs and an important characteristic of their carbon cycles. Copyright © 2015 John Wiley & Sons, Ltd.