Phosphorylation and dephosphorylation of the retinoblastoma protein, pRB, play a role in the control of cell cycle progression and expression of differentiation in eukaryotic cells. The regulation of pRB level and phosphorylation state was investigated during the induction of differentiation of murine erythroleukemia cells (MELC) by the chemical agent hexamethylene bisacetamide (HMBA). In MELC, there is a critical time in G1 or early S phase when HMBA must be present in order to induce differentiation. This is followed by prolongation of the subsequent G1 phase, resumption of progression through the cell cycle for several generations, and then cell cycle arrest in G1-G0. Associated with HMBA-induced prolongation of G1, there is an increase in the amount of the underphosphorylated form of pRB. A variant cell line (DS19/VCR-C) with accelerated kinetics of HMBA-mediated differentiation shows a more marked increase in underphosphorylated pRB. In culture with HMBA, as MELC resume progression through the cell cycle, pRB is present in the phosphorylated form. The total amount of pRB increases approximately 3-fold over the succeeding cell divisions prior to terminal arrest in G1. This increase in pRB is inhibited by dexamethasone, which also blocks HMBA-induced MELC differentiation. During this period, RB mRNA also increases approximately 3- to 5-fold, which reflects an increase in the rate of transcription, with no change in mRNA stability. The state of phosphorylation and amount of pRB appear to be involved in the control of HMBA-induced terminal cell division of MELC.