ABSTRACTProtoplasts were prepared from Ulva fasciata Delile, and their photosynthetic performance was measured and compared with that of thalli discs. These protoplasts maintained maximal rates of photosynthesis as high as those of thalli (up to 300 μmol O2·mg chlorophyll−1·h−1) for several hours after preparation and were therefore considered suitable for kinetic studies of inorganic carbon utilization. The photosynthetic K1/2(inorganic carbon) at pH 6.1 was 3.8 μM and increased to 67, 158, and 1410 μM at the pH values 7.0, 7.9, and 8.9, respectively. Compared with these protoplasts, thalli had a much lower affinity for CO2 but approximately the same affinity for HCO3−. Comparisons between rates of photosynthesis and the spontaneous dehydration of HCO3− (at 50 μM inorganic carbon) revealed that photosynthesis of both protoplasts (which lacked apparent activity of extracellular/surface‐bound carbonic anhydrase) and thalli (which were only 25% inhibited by the external carbonic anhydrase inhibitor acetazolamide) could not be supported by CO2 formation in the medium at the higher pH values, indicating HCO3− uptake. Since both protoplasts and thalli were sensitive to 4,4′‐diisothiocyanostilbene‐2,2′‐disulfonate, we suggest that HCO3− transport was facilitated by the membrane‐located anion exchange protein recently reported to function in certain Ulva thalli. These findings suggest that the presence of a cell wall may constitute a diffusion barrier for CO2, but not for HCO3−, utilization under natural seawater conditions.