SummaryToc64/OEP64 was identified biochemically in pea as a putative component of the chloroplast protein import apparatus. In Arabidopsis, three paralogous genes (atTOC64‐III,atTOC64‐VandatTOC64‐I) encode Toc64‐related proteins, and these have been reported to localize in chloroplasts, mitochondria and the cytosol, respectively. To assess the role of the atToc64‐III protein in chloroplast protein import in anin vivocontext, we identified and characterized Arabidopsis knockout mutants. The absence of detectable defects intoc64‐IIIsingle mutants raised the possibility of redundancy, and prompted us to also identifytoc64‐Vandtoc64‐Imutants, cross them totoc64‐III, and generate double‐ and triple‐mutant combinations. Thetoc64mutants were analysed carefully with respect to a variety of criteria, including chlorophyll accumulation, photosynthetic performance, organellar ultrastructure and chloroplast protein accumulation. In each case, the mutant plants were indistinguishable from wild type. Furthermore, the efficiency of chloroplast protein import was not affected by thetoc64mutations, even when a putative substrate of the atToc64‐III protein (wheatgerm‐translated precursor of the 33 kDa subunit of the oxygen‐evolving complex, OE33) was examined. Moreover, under various stress conditions (high light, osmotic stress and cold), thetoc64triple‐mutant plants were not significantly different from wild type. These results demonstrate that Toc64/OEP64 is not essential for the efficient import of proteins into chloroplasts in Arabidopsis, and draw into question the functional significance of this component.