Acute promyelocytic leukemia (APL) is characterized by the generation of the prototypic promyelocytic leukemia-retinoic acid receptor alpha (PML-RARα), an oncogenic fusion protein due to chromosomal translocation. In a human myeloid cell line, PML-RARα is cleaved by neutrophil elastase (NE) to produce the mutational PML [nuclear localization signal (NLS) deletion] and RARα (NLS-RARα, containing NLS of PML), both of which may play an important role in APL pathogenesis. The yeast two-hybrid technique was used to screen the intracellular proteins interacting with NLS-RARα, which may be involved in NLS-RARα signaling. The NLS-RARα coding sequence was amplified by polymerase chain reaction method and was cloned into the bait plasmid pGBKT7 vector, which, after the confirmation by sequencing, was transformed into yeast AH109 and the subsequent expression of bait plasmid was proved by Western-blot. The transformed yeast AH109 was mated with yeast YI87 (containing leukemia cDNA library plasmids pACT2) in medium. Diploid yeast was plated on synthetic dropout nutrient medium containing X-α-gal for screening. After being reintroduced into yeast AH109 and sequenced to verify the expression of ORF, eight positive colonies were obtained, among which one containing JTV-1 was cloned. The interaction between NLS-RARα and JTV-1 was further supported by indirect immunofluorescence, GST pull-down and co-immunoprecipitation, respectively. These findings brought some new clues for the further exploration of NLS-RARα signaling to APL.