Anaplerotic fixation of carbon dioxide by the fungus Aspergillus nidulans when grown under carbon-limited conditions was mediated by pyruvate carboxylase and a phosphoenol pyruvate (PEP)-metabolising enzyme which has been tentatively designated as PEP carboxylase. The activities of both enzymes were growth rate dependent and measurements of H14CO3 incorporation by growing mycelium indicated that they were responsible for almost all the assimilated carbon dioxide. In carbon-limited chemostats, the maximum rate of bicarbonate assimilation occurred at a dilution rate of 0.11 h-1, equivalent to 1/2 micromax. The affinity of the pyruvate carboxylase for bicarbonate was twice that of the PEP carboxylase under the conditions of growth used. The effect of changing the bicarbonate concentration in carbon-limited chemostats was substantial: increasing the HCO-3 concentration over the range 0.7 - 2.8 mM enhanced biomass synthesis by 22%. Over-shoots in bicarbonate assimilation and carboxylase activity occurred when steady state chemostat cultures were subjected to a step down in dilution rate.