Processes involved in photosynthetic CO2 acquisition were characterised for the isolated lichen photobiont Trebouxia erici (Chlorophyta, Trebouxiophyceae) and compared with Coccomyxa (Chlorophyta), a lichen photobiont without a photosynthetic CO2‐concentrating mechanism. Comparisons of ultrastructure and immuno‐gold labelling of ribulose‐1,5‐bisphosphate carboxylase‐oxygenase (Rubisco; EC 4.1.1.39) showed that the chloroplast was larger in T. erici and that the majority of Rubisco was located in its centrally located pyrenoid. Coccomyxa had no pyrenoid and Rubisco was evenly distributed in its chloroplast. Both species preferred CO2 rather than HCO3− as an external substrate for photosynthesis, but T. erici was able to use CO2 concentrations below 10–12 μM more efficiently than Coccomyxa. In T. erici, the lipid‐insoluble carbonic anhydrase (CA; EC 4.2.1.1) inhibitor acetazolamide (AZA) inhibited photosynthesis at CO2 concentrations below 1 μM, while the lipid‐soluble CA inhibitor ethoxyzolamide (EZA) inhibited CO2‐dependent O2 evolution over the whole CO2 range. EZA inhibited photosynthesis also in Coccomyxa, but to a much lesser extent below 10–12 μM CO2. The internal CA activity of Trebouxia, per unit chlorophyll (Chl), was ca 10% of that of Coccomyxa. Internal CA activity was also detected in homogenates from T. erici and two Trebouxia‐lichens (Lasallia hispanica and Cladina rangiferina). In all three, the predominating CA had α‐type characteristics and was significantly inhibited by low concentrations of AZA, having an I50 below 10–20 nM. In Coccomyxa a β‐type CA predominates, which is much less sensitive to AZA. Thus, the two photobionts differed in three major characteristics with respect to CO2 acquisition, the subcellular location of Rubisco, the relative requirement of CA and the biochemical characteristics of their predominating internal CA. These differences may be linked to the ability of Trebouxia to accumulate dissolved inorganic carbon internally, enhancing their CO2 use efficiency at and below air‐equilibrium concentrations (10–12 μM CO2) in comparison with Coccomyxa.