We investigated carbon acquisition by the N2-fixing cyanobacterium Trichodesmium IMS101 in response to CO2 levels of 15.1, 37.5, and 101.3 Pa (equivalent to 150, 370, and 1000 ppm). In these acclimations, growth rates as well as cellular C and N contents were measured. In vivo activities of carbonic anhydrase (CA), photosynthetic O2 evolution, and CO2 and HCO-3 fluxes were measured using membrane inlet mass spectrometry and the 14C disequilibrium technique. While no differences in growth rates were observed, elevated CO 2 levels caused higher C and N quotas and stimulated photosynthesis and N2 fixation. Minimal extracellular CA (eCA) activity was observed, indicating a minor role in carbon acquisition. Rates of CO2 uptake were small relative to total inorganic carbon (Ci) fixation, whereas HCO-3 contributed more than 90% and varied only slightly over the light period and between CO2 treatments. The low eCA activity and preference for HCO-3 were verified by the 14C disequilibrium technique. Regarding apparent affinities, half-saturation concentrations (K1/2) for photosynthetic O 2evolution and HCO-3 uptake changed markedly over the day and with CO2 concentration. Leakage (CO2 efflux : Ci uptake) showed pronounced diurnal changes. Our findings do not support a direct CO2 effect on the carboxylation efficiency of ribulose-1,5-bisphosphate carboxylase/oxygenase (RubisCO) but point to a shift in resource allocation among photosynthesis, carbon acquisition, and N 2 fixation under elevated CO2 levels. The observed increase in photosynthesis and N2 fixation could have potential biogeochemical implications, as it may stimulate productivity in N-limited oligotrophic regions and thus provide a negative feedback on rising atmospheric CO2 levels. © 2009, by the American Society of Limnology and Oceanography, Inc.