The 11.5-kDa zinc-binding protein (ZnBP, parathymosin-alpha), a potent inactivator of 1-phosphofructokinase, is found only in the cytoplasm of most tissues despite the presence of the putative nuclear localization signal PKRQKT. Recent reports on nuclear uptake of ZnBP could not exclude the participation of unspecific diffusion. We show here that wild-type ZnBP overexpressed in COS cells accumulates exclusively in the nucleus but that ZnBP with a mutated or deleted PKRQKT motif appears both in the nucleus and in the cytoplasm. In contrast, fusion proteins between ZnBP and parts of the endoplasmic reticulum protein calreticulin required the intact PKRQKT motif for nuclear import. The motif RKR, located nine amino acids upstream of the PKRQKT motif, is also involved in the active nuclear import of ZnBP. In contrast to rat hepatocytes and kidney cells in situ, which have ZnBP almost exclusively in the cytosol, we find ZnBP in Reuber H35 hepatoma cells and normal rat kidney cells only in the nuclei. Freshly isolated rat hepatocytes translocate their ZnBP to the nucleus in < 24 h during standard cell culture conditions.