Protein phosphatase type 1 (PP1) is involved in the regulation of numerous cell functions in mammalian cells. The major isoforms of PP1 catalytic subunit (PP1C)alpha, gamma1 and delta have nearly identical catalytic domain, but they vary in sequences at the amino and carboxyl termini. We previously showed that PP1Calpha is highly expressed in rat hepatoma cells. To examine isoform specific function of PP1C, each isoform was depleted from HeLa cells by RNA interference. The PP1Calpha-depleted cells rounded up and showed increased cell death, indicating that PP1Calpha is essential in cell proliferation. PP1Cgamma1-depleted cells slightly rounded up and have decreased G1 phase population and increased S phase population. The PP1Cdelta-depleted cells were enlarged, and appeared flat and rich in lamellipodia. These data suggested that each PP1C isoform has non-redundant function in vivo.