In July–September 2007 at monthly intervals, we performed four experiments on size-fractionated phytoplankton organic carbon uptake in the eutrophic Římov Reservoir (Czech Republic). Along with primary production measurement, samples from surface and euphotic depth were incubated at in situ depths in light and dark bottles with ³H-labelled amino acid mixture and glucose in nanomolar concentrations. Moreover, we measured silica deposition and chlorophyll content in diatom cells with and without unlabelled glucose addition (100 µM). Phytoplankton uptake of both glucose and amino acids was low regardless of the incubation depth. The major portion of incorporated organic carbon was found in the fraction <2 µm indicating that bacteria are better competitors for organic substrate than phytoplankton. Additionally, direct visualization of organic carbon uptake using microautoradiography showed that bacteria attached to the phytoplankton accounted for the activity in the large size fractions. Thus, mixotrophic nutrition of the phytoplankton was of low importance under natural conditions. However, glucose addition in micromolar concentrations resulted in a significant increase in diatom silification rates in the light bottles. Interestingly, in both light and dark bottles, chlorophyll fluorescence in the diatom cells markedly decreased after the glucose addition indicating a potential restructuring of cell metabolism to benefit from a remarkably high concentration of available organic carbon in the environment.