Maize chloroplast protein synthesis elongation factor, EF-Tu, has been implicated in heat tolerance, and previous studies have shown that under heat stress this protein accumulates in 14-d-, 17-d-, and 21-d-old plants of maize genotypes with increased tolerance to stress. In the present study, we investigated the expression of EF-Tu genes in heat tolerant, ZPBL 1304, and heat sensitive, ZPL 389, maize lines during early stages of their development (5-21-d-old plants) under both control and heat stress conditions. We also investigated the expression of EF-Tu in mature plants of these lines under field conditions and assessed heat tolerance in young seedlings at different stages of their development. The expression of EF-Tu was studied by determining the relative levels of EF-Tu protein and the steady state levels of EF-Tu mRNA. Chloroplast EF-Tu showed differential expression during early stages of plant development, and the heat tolerant and the heat sensitive line differed in the expression of EF-Tu under heat stress. In ZPBL 1304, plants of all ages (except 5-d-old shoots) showed heat-induced accumulation of both EF-Tu transcript and EF-Tu protein. In contrast, in ZPL 389, only plants up to 14d of age displayed increased accumulation of EF-Tu under heat stress. The increase in the relative level of EF-Tu in ZPL 389 was not preceded by an increase in the steady state level of EF-Tu mRNA. Under heat stress, the relative levels of EF-Tu correlated positively with plant heat tolerance. The results are consistent with the hypothesis that maize EF-Tu plays a role in heat tolerance and suggest that under heat stress conditions, the regulation of expression of EF-Tu may be different in the heat tolerant and heat sensitive maize lines.