The light activation of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) in vivo requires the presence of Rubisco activase, a nuclear-encoded chloroplast protein that consists of two isoforms arising from alternative splicing in most plants. We examined the function of each isoform by characterizing Rubisco activation in transgenic Arabidopsis plants that express only one or both isoforms, as compared with the wild type. In plants expressing only the shorter isoform, Rubisco activity was as high as in the wild type under saturating light, but the activity was not down-regulated at intensities limiting for photosynthesis. In contrast, in plants expressing only the longer isoform, Rubisco activity was down-regulated at limiting light, but the activity was slightly lower and increased much more slowly at saturating light intensities as compared with the wild type. Light regulation of Rubisco similar to that in the wild-type plants was observed in the progeny of a genetic cross of these two transformants in which both isoforms were again present. When the capacity to redox regulate the activity of the larger activase isoform was eliminated by replacement of the critical cysteine residues in the carboxyl-terminal extension unique to this isoform, Rubisco activity in saturating light was similar to the wild type, but the ability of the larger isoform to down-regulate Rubisco activity at limiting light intensities in transgenic plants was almost abolished. These results indicate that the light modulation of Rubisco under limiting light is mainly due to the ability to regulate the activity of Rubisco activase by redox changes in the stroma.