Phosphorylation of the protein encoded by retinoblastoma susceptibility gene (pRb) is the key event of the cell cycle committing the cell to enter S phase and also required for progression through S and G2. We describe a new methodology to monitor pRb phosphorylation in individual cells and correlate it with the cell cycle position. Specifically, pRb phosphorylation in human lymphocytes was assayed immunocytochemically using mAb which recognizes underphosphorylated pRb (pRbP-) conjugated with a fluorochrome of one color combined with mAb which reacts with pRb regardless of its phosphorylation (total pRb; pRbT) tagged with another color fluorochrome. DNA was stained with still another color fluorochrome and cell fluorescence was measured by multiparameter flow cytometry. Specificity of anti-pRbP- mAb was confirmed by preincubation of the permeabilized cells with phosphatase. Analysis of pRbP- or a ratio of pRbP-/pRbT revealed that pRb was underphosphorylated in over 98% of the nonstimulated lymphocytes. The proportion of cells with underphosphorylated pRb dropped to 20% between 3 and 8 h after addition of the mitogen phytohemagglutinin (PHA). Phosphorylation of pRb within a cell was rapid and complete since reactivity of individual lymphocytes with anti-pRbP-mAb was lost abruptly rather than step-wise during stimulation. Phosphorylation of pRb coincided with the appearance of cyclin D3, which was induced 3 h and peaked 12 h after addition of PHA. The nonspecific protein kinase inhibitor staurosporine at a concentration known to arrest lymphocytes in G1 but not to interfere with the induction of cyclin D3 (20 nM) prevented pRb phosphorylation. The present assay can be applied for screening antitumor drugs targeting CDKs and be useful for monitoring pRb phosphorylation in human tumors, the feature of a possible prognostic value in oncology.