Protein Ser/Thr phosphatase-1 (PP1) is a ubiquitous eukaryotic enzyme that controls numerous cellular processes by the dephosphorylation of key regulatory proteins. PP1 is expressed in various cellular compartments but is most abundant in the nucleus. We have examined the determinants for the nuclear localization of enhanced green fluorescent protein-tagged PP1 in COS1 cells. Our studies show that PP1gamma(1) does not contain a functional nuclear localization signal and that its nuclear accumulation does not require Sds22, which has previously been implicated in the nuclear accumulation of PP1 in yeast (Peggie, M. W., MacKelvie, S. H., Bloecher, A., Knatko, E. V., Tatchell, K., and Stark, M. J. R. (2002) J. Cell Sci. 115, 195-206). However, the nuclear targeting of PP1 isoforms was alleviated by the mutation of their binding sites for proteins that interact via an RVXF motif. Moreover, one of the mutants with a cytoplasmic accumulation and decreased affinity for RVXF motifs (PP1gamma(1)-F257A) could be re-targeted to the nucleus by the overexpression of nuclear interactors (NIPP1 (nuclear inhibitor of PP1) and PNUTS (PP1 nuclear targeting subunit)) with a functional RVXF motif. Also, the addition of a synthetic RVXF-containing peptide to permeabilized cells resulted in the loss of nuclear enhanced green fluorescent protein-PP1gamma(1). Finally, NIPP1(-/-) mouse embryos showed a nuclear hyperphosphorylation on threonine, consistent with a role for NIPP1 in the nuclear targeting and/or retention of PP1. Our data suggest that both the nuclear translocation and the nuclear retention of PP1 depend on its binding to interactors with an RVXF motif.