The role of HSDJ, a human homolog of bacterial DnaJ and yeast YDJ1p/MAS5, in mitochondrial protein import was examined. Recombinant HSDJ was purified and an antibody was prepared. HSDJ mRNA was heat-induced in cultured cells. In pulse-labeling and chase experiments using COS-7 cells, the endogenous HSDJ homolog was prenylated. Transiently expressed HSDJ was also prenylated, whereas its mutant C394S in which cysteine of the "CaaX box" was mutated to serine, was not. HSDJ, but not C394S, synthesized in rabbit reticulocyte lysate was farnesylated. The HSDJ antibody inhibited import of ornithine transcarbamylase precursor (pOTC) into isolated mitochondria when added prior to pOTC synthesis, but not when added prior to import assay. In transient expression of pOTC in COS-7 cells, pOTC was synthesized and processed to the mature form with an apparent half-life of 2-3 min. Coexpression of HSDJ or C394S resulted in slight retardation of the pOTC processing. These results indicate that HSDJ is involved in an early step(s) of protein import into mitochondria.