The basis of inhibition of photosynthesis by single acute O(3) exposures was investigated in vivo using analyses based on leaf gas exchange measurements. The fully expanded second leaves of wheat plants (Triticum aestivum L. cv Avalon) were fumigated with either 200 or 400 nanomoles per mole O(3) for between 4 and 16 hours. This reduced significantly the light-saturated rate of CO(2) uptake and was accompanied by a parallel decrease in stomatal conductance. However, the stomatal limitation, estimated from the relationship between CO(2) uptake and the internal CO(2) concentration, only increased significantly during the first 8 hours of exposure to 400 nanomoles per mole O(3); no significant increase occurred for any of the other treatments. Analysis of the response of CO(2) uptake to the internal CO(2) concentration implied that the predominant factor responsible for the reduction in light-saturated CO(2) uptake was a decrease in the efficiency of carboxylation. This was 58 and 21% of the control value after 16 hours at 200 and 400 nanomoles per mole O(3), respectively. At saturating concentrations of CO(2), photosynthesis was inhibited by no more than 22% after 16 hours, indicating that the capacity for regeneration of ribulose bisphosphate was less susceptible to O(3). Ozone fumigations also had a less pronounced effect on light-limited photosynthesis. The maximum quantum yield of CO(2) uptake and the quantum yield of oxygen evolution showed no significant decline after 16 hours with 200 nanomoles per mole O(3), requiring 8 hours at 400 nanomoles per mole O(3) before a significant reduction occurred. The photochemical efficiency of photosystem II estimated from the ratio of variable to maximum chlorophyll fluorescence and the atrazine-binding capacity of isolated thylakoids demonstrated that photochemical reactions were not responsible for the initial inhibition of CO(2) uptake. The results suggest that the apparent carboxylation efficiency appears to be the initial cause of decline in photosynthesis in vivo following acute O(3) fumigation.