The Ycf3 protein is essential for the accumulation of the photosystem I (PSI) complex and acts at a post-translationa level. The sequence of Ycf3 is conserved in cyanobacteria, algae, and plants and contains three tetratrico-peptide re? peats (TPR). TPRs have been shown to function as sites for protein-protein interactions. The mutations Y95A/Y96A Y142A/W143A in the second and third TPR repeats lead to a modest decrease of PSI, but they prevent photoau- totrophic growth and cause enhanced light sensitivity even though the accumulated PSI complex is fully functional. This phenotype can be reversed under anaerobic conditions and appears to be the result of photooxidative damage. A temperature-sensitive ycf3 mutant, generated by random mutagenesis of a conserved region near the N-terminal end of Ycf3, was used in temperature-shift experiments to show that Ycf3 is required for PSI assembly but not for its stabil? ity. Immunoblot analysis of thylakoid membranes separated by two-dimensional gel electrophoresis and immunopre- cipitations shows that Ycf3 interacts directly with the PSI subunits PsaA and PsaD, but not with subunits from other photosynthetic complexes. Thus, Ycf3 appears to act as a chaperone that interacts directly and specifically with at least two of the PSI subunits during assembly of the PSI complex.