The cell cycle phase-specific phosphorylation status of the RB protein (retinoblastoma tumor suppressor gene product) during an elicited cellular program of G0 arrest and cell differentiation was characterized. The RB protein phosphorylation state is presumed to be an important determinant of cell proliferation or arrest. The cell cycle phase specificity of RB protein phosphorylation and dephosphorylation during HL-60 human leukemia cell proliferation and differentiation was determined using DNA-based fluorescence-activated cell sorting and Western analysis. The RB protein in proliferating G1 cells was phosphorylated, but at a relatively low level. The extent of phosphorylation increased in S phase cells and was maximum in G2 + M cells. After the cells were treated with retinoic acid or 1,25-dihydroxy vitamin D3, they began to accumulate in G1/0 and phenotypically convert. Significant unphosphorylated RB protein did not appear until after the first cells had arrested and differentiated. Dephosphorylation of the RB protein was first apparent at the beginning of G2 in the remaining cycling cells after onset of arrest and differentiation had already occurred. By the time the remaining cycling cells had divided and arrested, resulting in G0 cells, a majority of RB protein was dephosphorylated, but some remained phosphorylated. The data indicate that dephosphorylation of RB does not determine residence in G1/0. Rather dephosphorylation appears as one relatively late-occurring component of the metabolic cascade culminating in G0-arrested, phenotypically differentiated cells. Dephosphorylated RB appears as a feature of differentiated cells. The data are consistent with a role for hypophosphorylated RB not so much in deriving, but in possibly sustaining the differentiated state.