Carbonic anhydrase (CA, EC 188.8.131.52) catalyses the first reaction in the C4 photosynthetic pathway, the conversion of atmospheric CO2 to bicarbonate in the mesophyll cytosol. To examine the importance of the enzyme to the functioning of the C4 photosynthetic pathway, Flaveria bidentis (L.) Kuntze, a C4 dicot, was genetically transformed with an antisense construct in which the cDNA encoding a putative cytosolic CA (CA3) was placed under the control of a constitutive promoter. Some of the primary transformants had impaired CO2 assimilation rates and required high CO2 for growth. The T1 progeny of four primary transformants were used to examine the quantitative relationship between leaf CA activity and CO2 assimilation rate. CA activity was determined in leaf extracts with a mass spectrometric technique that measured the rate of 18O exchange from doubly labelled 13C18O2. Steady-state CO2 assimilation rates were unaffected by a decrease in CA activity until CA activity was less than 20% of wild type when they decreased steeply. Transformants with less than 10% of wild-type CA activity had very low CO2 assimilation rates and grew poorly at ambient CO2 partial pressure. Reduction in CA activity also increased the CO2 partial pressure required to saturate CO2 assimilation rates. The present data show that CA activity is essential for the functioning of the C4 photosynthetic pathway.