DJA1 and DJA2 are Hsp40-family co-chaperone proteins that regulate the activity of the chaperone Hsc70/Hsp70. The N-terminal J domains of DJA1 and DJA2 promote ATP hydrolysis and polypeptide binding by Hsc70. Both co-chaperones are also thought to bind unfolded polypeptides in their central to C-terminal regions. Yet, the two are functionally distinct, possibly due to divergence in the connections between their functional domains. Here, we constructed C-terminal fragments of DJA1 (A1-C, residues 254-397) and DJA2 (A2-C, residues 254-412), expected to contain the homodimerization site but not the main polypeptide-binding region. The fragments were expressed in E. coli under optimized conditions, and purified. Co-precipitation experiments with the pure fragments suggested that A1-C retained partial binding to model polypeptides, while A2-C showed less binding. The fragments were then transfected into HeLa cells, in which overexpression of full-length DJA1 and DJA2 had previously been found to increase the folding of a co-transfected model protein. Both A1-C and A2-C increased folding moderately, less than the full-length co-chaperones, but notably the fragments did not inhibit folding. These results suggest that these C-terminal domains may provide a second site for polypeptide binding, contributing to the substrate specificity or overall activity of the co-chaperones.