In eukaryotes, proteins homologous to the bacterial DnaJ protein are involved in regulation of the Hsp70 molecular chaperones, which are implicated in a variety of protein biogenesis pathways. We report herewith the molecular characterization of a T. cruzi DnaJ gene, termed TcJ6, encoding a protein that displays high sequence homology with the Saccharomyces cerevisiae Sis1 co-chaperone required for the initiation of translation. TcJ6 protein was expressed as a polypeptide of 36.5 kDa at a constant level during parasite differentiation and was associated to the cytoplasmic fraction. We showed that overexpression of TcJ6 complemented a temperature-sensitive yeast sis1 mutant. In addition, sucrose gradient sedimentation analysis of polysomes from T. cruzi and a yeast mutant overexpressing TcJ6p showed that the trypanosomal co-chaperone was closely associated with ribosomal subunits, 80 S monosomes and the smaller polysomes, as observed for Sis1p. Furthermore, in T. cruzi TcJ6p was also found to be preferentially concentrated around the nucleus, giving a speckled staining pattern. This suggests that TcJ6p is associated with the endoplasmic reticulum. Taken together, these data suggest that the trypanosomal DnaJ is involved in initiation of translation.