Chloroplast protein synthesis elongation factor, EF-Tu, has been implicated in heat tolerance in maize. The recombinant precursor of this protein, pre-EF-Tu, has been found to exhibit chaperone activity and protect heat-labile proteins, such as citrate synthase and malate dehydrogenase, from thermal aggregation. Chloroplast EF-Tu is highly conserved and it is possible that the chaperone activity of this protein is not species-specific. In this study, we investigated the effect of native wheat pre-EF-Tu on thermal aggregation of rubisco activase. Additionally, we investigated the effect of native and recombinant maize pre-EF-Tu on activase aggregation. Activase was chosen because it displays an exceptional sensitivity to thermal aggregation and constrains photosynthesis at high temperature. The native precursors of both wheat and maize EF-Tu displayed chaperone activity, as shown by the capacity of both proteins to reduce thermal aggregation of rubisco activase in vitro. Similarly, the recombinant maize pre-EF-Tu protected activase from thermal aggregation. This is the first report on chaperone activity of native pre-EF-Tu and the first evidence for thermal protection of a photosynthetic enzyme by this putative chaperone. The results are consistent with the hypothesis that chloroplast EF-Tu plays a functional role in heat tolerance by acting as a molecular chaperone.