The activation of Rubisco in vivo requires the presence of the regulatory protein Rubisco activase. This enzyme facilitates the release of sugar phosphate inhibitors from Rubisco catalytic sites thereby influencing carbamylation. T(1) progeny of transgenic Flaveria bidentis (a C(4) dicot) containing genetically reduced levels of Rubisco activase were used to explore the role of the enzyme in C(4) photosynthesis at high temperature. A range of T(1) progeny was screened at 25 degrees C and 40 degrees C for Rubisco activase content, photosynthetic rate, Rubisco carbamylation, and photosynthetic metabolite pools. The small isoform of F. bidentis activase was expressed and purified from E. coli and used to quantify leaf activase content. In wild-type F. bidentis, the activase monomer content was 10.6+/-0.8 micromol m(-2) (447+/-36 mg m(-2)) compared to a Rubisco site content of 14.2+/-0.8 micromol m(-2). CO(2) assimilation rates and Rubisco carbamylation declined at both 25 degrees C and 40 degrees C when the Rubisco activase content dropped below 3 mumol m(-2) (125 mg m(-2)), with the status of Rubisco carbamylation at an activase content greater than this threshold value being 44+/-5% at 40 degrees C compared to 81+/-2% at 25 degrees C. When the CO(2) assimilation rate was reduced, ribulose-1,5-bisphosphate and aspartate pools increased whereas 3-phosphoglycerate and phosphoenol pyruvate levels decreased, demonstrating an interconnectivity of the C(3) and C(4) metabolites pools. It is concluded that during short-term treatment at 40 degrees C, Rubisco activase content is not the only factor modulating Rubisco carbamylation during C(4) photosynthesis.