In eukaryotes, from fly to human, nine aminoacyl-tRNA synthetases contribute a multienzyme complex of defined and conserved structural organization. This ubiquitous multiprotein assemblage comprises a unique bifunctional aminoacyl-tRNA synthetase, glutamyl-prolyl-tRNA synthetase, as well as the monospecific isoleucyl, leucyl, glutaminyl, methionyl, lysyl, arginyl, and aspartyl-tRNA synthetases. Three auxiliary proteins of apparent molecular masses of 18, 38 and 43 kDa are invariably associated with the nine tRNA synthetase components of the complex. As part of an inquiry into the molecular and functional organization of this macromolecular assembly, we isolated the cDNA encoding the p38 non-synthetase component and determined its function. The 320 amino acid residue encoded protein has been shown to have no homolog in yeast, bacteria and archaea, according to the examination of the complete genomic sequences available. The p38 protein is a moderately hydrophobic protein, displays a putative leucine-zipper motif, and shares a sequence pattern with protein domains that are involved in protein-protein interactions. We used the yeast two-hybrid system to register protein connections between components of the complex. We performed an exhaustive search of interactive proteins, involving 10 of the 11 components of the complex. Twenty-one protein pairs have been unambiguously identified, leading to a global view of the topological arrangement of the subunits of the multisynthetase complex. In particular, p38 was found to associate with itself to form a dimer, but also with p43, with the class I tRNA synthetases ArgRS and GlnRS, with the class II synthetases AspRS and LysRS, and with the bifunctional GluProRS. We generated a series of deletion mutants to localize the regions of p38 mediating the identified interactions. Mapping the interactive domains in p38 showed the specific association of p38 with its different protein partners. These findings suggest that p38, for which no homologous protein has been identified to date in organisms devoid of multisynthetase complexes, plays a pivotal role for the assembly of the subunits of the eukaryotic tRNA synthetase complex.