Thylakoid membrane-localized chloroplast ATP synthases use the proton motive force generated by photosynthetic electron transport to produce ATP from ADP. Although it is well known that the chloroplast ATP synthase is composed of more than 20 proteins with α₃β₃γ₁ε₁δ₁I₁II₁III₁₄IV₁ stoichiometry, its biogenesis process is currently unclear. To unravel the molecular mechanisms underlying the biogenesis of chloroplast ATP synthase, we performed extensive screening for isolating ATP synthase mutants in Arabidopsis (Arabidopsis thaliana). In the recently identified bfa3 (biogenesis factors required for ATP synthase 3) mutant, the levels of chloroplast ATP synthase subunits were reduced to approximately 25% of wild-type levels. In vivo labeling analysis showed that assembly of the CF₁ component of chloroplast ATP synthase was less efficient in bfa3 than in the wild type, indicating that BFA3 is required for CF₁ assembly. BFA3 encodes a chloroplast stromal protein that is conserved in higher plants, green algae, and a few species of other eukaryotic algae, and specifically interacts with the CF₁β subunit. The BFA3 binding site was mapped to a region in the catalytic site of CF₁β. Several residues highly conserved in eukaryotic CF₁β are crucial for the BFA3–CF₁β interaction, suggesting a coevolutionary relationship between BFA3 and CF₁β. BFA3 appears to function as a molecular chaperone that transiently associates with unassembled CF₁β at its catalytic site and facilitates subsequent association with CF₁α during assembly of the CF₁ subcomplex of chloroplast ATP synthase.