The spatial variability in carbon dioxide in surface waters of a coastal area of the Gulf of Cádiz (southwestern Spain) was examined in situ under spring bloom conditions. The influence of phytoplankton biomass and physicochemical variables on the CO2 concentration was studied. According to the relationship observed between chlorophyll a and pCO2, phytoplankton biomass was the main factor responsible for variations in carbon dioxide. The distribution of organic matter in the form of dissolved organic carbon and transparent exopolymer particles also reflected changes in phytoplankton abundance, since high levels of both variables were associated with high chlorophyll concentrations and low levels of free CO2. The involvement of the enzyme carbonic anhydrase in the process of inorganic carbon uptake by the phytoplankton community was also investigated through the effect of the inhibitors dextran-bound sulfonamide and ethoxyzolamide on primary production rates. Ethoxyzolamide substantially inhibited carbon fixation, causing decreases of 40%–60% in the maximum photosynthetic rates; whereas the membrane impermeable inhibitor dextran-bound sulfonamide affected primary production, depending on the diversity of the phytoplankton species composition. Calculations of CO2 fluxes indicated that the sampled coastal sector of the Gulf of Cádiz behaved as a net sink for atmospheric CO2 at the time of analysis, with an average CO2 absorption of –0.41 mmol·m–2·d–1.Key words: air–sea exchange of CO2, carbonic anhydrase, flow cytometry, Gulf of Cádiz, phytoplankton, transparent exopolymer particles.