We have previously reported on the M-phase specific dephosphorylation of pRb and identified a type 1 serine/threonine protein phosphatase (PP1) as the enzyme mediating pRb dephosphorylation. In this report, we have characterized the pRb-directed phosphatase activity found in mitotic cells with respect to dose dependence and demonstrate that the pRb isoform conversion detected in vitro mirrors the pRb isoform conversion which occurs during mitosis of intact cells. Cell fractionation and PP1 catalytic subunit isolation studies support the notion that the pRb-directed phosphatase activity involves subpopulations of PP1 catalytic subunits. Coprecipitation studies revealed that PP1 can form a complex with hypophosphorylated pRb which was converted from the hyperphosphorylated form in mitotic cell extracts. Taken together with data from previous reports in the literature, a model for the regulation of PP1 activity towards pRb during mitotic exit is proposed.