In freshwaters, algal species are exposed to different inorganic nitrogen (Ni) sources whose incorporation varies in biochemical energy demand. We hypothesized that due to the lesser energy requirement of ammonium (NH4+)-use, in contrast to nitrate (NO3-)-use, more energy remains for other metabolic processes, especially under CO2- and phosphorus (Pi) limiting conditions. Therefore, we tested differences in cell characteristics of the green alga Chlamydomonas acidophila grown on NH4+ or NO3- under covariation of CO2 and Pi-supply in order to determine limitations, in a full-factorial design. As expected, results revealed higher carbon fixation rates for NH4+-grown cells compared to growth with NO3- under low CO2 conditions. NO3--grown cells accumulated more of the nine analysed amino acids, especially under Pi-limited conditions, compared to cells provided with NH4+. This is probably due to a slower protein synthesis in cells provided with NO3-. In contrast to our expectations, compared to NH4+-grown cells NO3--grown cells had higher photosynthetic efficiency under Pi-limitation. In conclusion, growth on the Ni-source NH4+ did not result in a clearly enhanced Ci-assimilation, as it was highly dependent on Pi and CO2 conditions (replete or limited). Results are potentially connected to the fact that C. acidophila is able to use only CO2 as its inorganic carbon (Ci) source.