Elevated CO₂ leads to a decrease in potential net photosynthesis in long-term experiments and thus to a reduction in potential growth. This process is known as photosynthetic downregulation. There is no agreement on the definition of which parameters are the most sensitive for detecting CO₂ acclimation. In order to investigate the most sensitive photosynthetic and molecular markers of CO₂ acclimation, the effects of elevated CO₂, and associated elevated temperature were analyzed in alfalfa plants inoculated with different Sinorhizobium meliloti strains. Plants (Medicago sativa L. cv. AragÓn) were grown in summer or autumn in temperature gradient greenhouses (TGG). At the end of the experiment, all plants showed acclimation in both seasons, especially under elevated summer temperatures. This was probably due to the lower nitrogen (N) availability caused by decreased N₂-fixation under higher temperatures. Photosynthesis measured at growth CO₂ concentration, rubisco in vitro activity and maximum rate of carboxylation were the most sensitive parameters for detecting downregulation. Severe acclimation was also related with decreases in leaf nitrogen content associated with declines in rubisco content (large and small subunits) and activity that resulted in a drop in photosynthesis. Despite the sensitivity of rubisco content as a marker of acclimation, it was not coordinated with gene expression, possibly due to a lag between gene transcription and protein translation.