Carbonic anhydrase (CA, EC 22.214.171.124) catalyses the firstreaction in the C4photosynthetic pathway, the conversionof atmospheric CO2to bicarbonate in the mesophyll cytosol.To examine the importance of the enzyme to the functioningof the C4photosynthetic pathway,Flaveria bidentis(L.) Kuntze, a C4dicot, was genetically transformed withan antisense construct in which the cDNA encoding a putativecytosolic CA (CA3) was placed under the control of aconstitutive promoter. Some of the primary transformantshad impaired CO2assimilation rates and required high CO2for growth. The T1progeny of four primary transformantswere used to examine the quantitative relationship betweenleaf CA activity and CO2assimilation rate. CA activity wasdetermined in leaf extracts with a mass spectrometric techniquethat measured the rate of18O exchange from doublylabelled13C18O2. Steady-state CO2assimilation rates wereunaffected by a decrease in CA activity until CA activitywas less than 20% of wild type when they decreased steeply.Transformants with less than 10% of wild-type CA activityhad very low CO2assimilation rates and grew poorly atambient CO2partial pressure. Reduction in CA activityalso increased the CO2partial pressure required to saturateCO2assimilation rates. The present data show that CAactivity is essential for the functioning of the C4photosyntheticpathway.