RE-1 silencer transcription factor (REST), also known as neuron-restrictive silencer factor (NRSF), contains nine Cys2-His2 type zinc finger domains (ZFDs). REST/NRSF is localized to the nucleus, where it represses the transcriptional activity of a large number of neuronal genes in non-neuronal cells. It has been suggested that REST/NRSF contains a nuclear localization signal (NLS) corresponding to amino acids (512-522). However, our studies showed that REST4, a REST/NRSF splicing isoform, which contains the N-terminal 5 of 9 ZFDs, efficiently localized to the nucleus. On the other hand REST1, another REST/NRSF splicing isoform, which contains 4 of the 9 ZFDs, localized to the cytosol. In this study REST-DeltaC, which contains 8 ZFDs with the NLS (512-522) deleted, was found to localize to the nucleus in HeLa, COS and PC12 cells. Complete deletion or mutation of NLS (512-522) still permitted REST/NRSF to be localized to the nucleus in HeLa, COS and PC12 cells. In contrast REST/NRSF constructs which contain a deletion of ZFD-5 mislocalized to the cytosol. A point mutation in the zinc finger structure that disrupts its conformation remains nuclear. These data suggest that REST/NRSF contains a NLS around ZFD-5, while the putative NLS at residues 512-522 is non-functional.