DnaJ is an essential cochaperone of mammalian heat shock cognate 70 (hsc70) protein. We previously found that dj2 (HSDJ/hdj-2/rdj1), rather than dj1 (hsp40/hdj-1), is a partner DnaJ for the hsc70-based chaperone system. Here, we compared the distribution of dj1, dj2, and the newly found dj3 (cpr3/DNJ3/HIRIP4/rdj2) in cultured cells. Both dj3 as well as dj2 were farnesylated and were ubiquitously expressed. In immunocytochemical and subfractionation studies, these two proteins colocalized with hsc70 under normal conditions. However, dj1 and hsc70 apparently colocalized in the nucleoli after heat shock. Simultaneous depletion of dj2 and dj3 from rabbit reticulocyte lysate markedly reduced mitochondrial import of pre-ornithine transcarbamylase and refolding of guanidine-denatured luciferase. Re-addition of either dj2 or dj3 led to recovery of these reactions. In a reconstituted system, both hsc70-dj2 and hsc70-dj3 were effective in protein refolding. Anti-apoptotic protein bag-1 further stimulated ATP hydrolysis and protein refolding by both pairs. Thus, dj2 and dj3 are the partner DnaJs of hsc70 within the cell, functionally similar and much more efficient than dj1, and bag-1 is a positive cochaperone of the hsc70-dj2 and hsc70-dj3 systems.