Protein phosphatase 1 (PP1) is a ubiquitous serine/threonine phosphatase that regulates many cellular processes, including cell division. When transiently expressed as fluorescent protein (FP) fusions, the three PP1 isoforms, α, β/δ, and γ1, are active phosphatases with distinct localization patterns. We report here the establishment and characterization of HeLa cell lines stably expressing either FP-PP1γ or FP alone. Time-lapse imaging reveals dynamic targeting of FP-PP1γ to specific sites throughout the cell cycle, contrasting with the diffuse pattern observed for FP alone. FP-PP1γ shows a nucleolar accumulation during interphase. On entry into mitosis, it localizes initially at kinetochores, where it exchanges rapidly with the diffuse cytoplasmic pool. A dramatic relocalization of PP1 to the chromosome-containing regions occurs at the transition from early to late anaphase, and by telophase FP-PP1γ also accumulates at the cleavage furrow and midbody. The changing spatio-temporal distribution of PP1γ revealed using the stable PP1 cell lines implicates it in multiple processes, including nucleolar function, the regulation of chromosome segregation and cytokinesis.