Previously, we characterized a mitochondrial co-chaperonin (Cpn10) and a chloroplast co-chaperonin (Cpn20) from Arabidopsis thaliana (Koumoto, Y., Tsugeki, R., Shimada, T., Mori, H., Kondo, M., Hara-Nishimura, I., and Nishimura, M. (1996) Plant J. 10, 1119-1125; Koumoto, Y., Shimada, T., Kondo, M., Takao, T., Shimonishi, Y., Hara-Nishimura, I., and Nishimura, M. (1999) Plant J. 17, 467-477). Here, we report a third co-chaperonin. The cDNA was 603 base pairs long, encoding a protein of 139 amino acids. From a sequence analysis, the protein was predicted to have one Cpn10 domain with an amino-terminal extension that might work as a chloroplast transit peptide. This novel Cpn10 was confirmed to be localized in chloroplasts, and we refer to it as chloroplast Cpn10 (chl-Cpn10). The phylogenic tree that was generated with amino acid sequences of other co-chaperonins indicates that chl-Cpn10 is highly divergent from the others. In the GroEL-assisted protein folding assay, about 30% of the substrates were refolded with chl-Cpn10, indicating that chl-Cpn10 works as a co-chaperonin. A Northern blot analysis revealed that mRNA for chl-Cpn10 is accumulated in the leaves and stems, but not in the roots. In germinating cotyledons, the accumulation of chl-Cpn10 was similar to that of chloroplastic proteins and accelerated by light. It was proposed that two kinds of co-chaperonins, Cpn20 and chl-Cpn10, work independently in the chloroplast.