We have examined the effect of inorganic and organic carbon nutrition on the physiological expression of HCO3- transport and the CO2-concentrating mechanism (CCM) in the nutritionally versatile cyanobacterium Chlorogloeopsis sp. ATCC 27193. Cells grown under photoautotrophic conditions in the presence of limiting or replete levels of inorganic carbon (Ci), or grown under mixotrophic (light) or chemoheterotrophic (dark) conditions in the presence of sucrose retained both active CO2 and Na(+)-independent HCO3- transport activity. However, two distinct effects on the kinetic properties of HCO3- transport were observed, which segregated on the basis of phototrophic and chemoheterotrophic growth in the dark. In the former, the apparent substrate affinity of the HCO3- transport system (K0.5) varied (12-fold) in response to the growth Ci or mixotrophy while the maximum rate of HCO3- transport was approximately constant. In the latter case, the K0.5 value was unchanged from the starting value (35 microM) of Ci-limited photoautotrophic cells used to initiate the dark-grown cultures, but transport capacity declined 3-fold. Modulation of the K0.5 (HCO3- transport) value required light. Cellular carboxysome content was unaffected by growth under any of the regimes employed and these structures were the predominant location of ribulose-1,5-bisphosphate carboxylase/oxygenase, as indicated by immunogold electron microscopy. Mixotrophic and chemoheterotrophic growth resulted in a diminished ability to concentrate Ci internally and a reduction in Ci accumulation ratios at low external Ci concentrations. The relationship between photosynthetic carbon fixation and the internal Ci pool varied by 2-fold, with high-Ci-grown cells being the most efficient and mixotrophically grown cells the least, indicating that there was limited capacity to modulate this relationship in response to changes in carbon nutrition. Within broad limits this relationship appeared to be a fixed trait of the strain and an important factor in determining growth rate.