A 939-amino acid monomeric class I tRNA synthetase has been split into three inactive peptides. The three peptides spontaneously assemble in vivo to reconstitute active protein. Active tripartite complexes were demonstrated in vitro. The tripartite assembly of this synthetase increases by several-fold the size of a polypeptide that has been demonstrated to be assembled from more than two constituent pieces. The results indicate that contemporary single-chain tRNA synthetases or other large proteins could in principle develop from intermediates composed of non-covalent assemblages of multiple peptides.