Immunosuppressive agents may initiate their pharmacologic action by disrupting phosphorylation cascades critical to T lymphocyte intracellular signaling after alloantigen recognition. Activator of DNA Replication (ADR), a transduction signal sensitive to CsA as well as rapamycin (RAPA) immunosuppression, seemed a likely candidate for phosphoregulation. This communication reports the development of a cell-free assay wherein CsA and RAPA inhibit ADR induction by protein kinase C (PKC). ADR and PKC are inactive in the cytosol of resting cells. Endogenous PKC activity in quiescent lymphocytes was triggered with the tumor promoter PMA, leading to the appearance of ADR, an event subsequently quantitated by the ability of ADR to trigger [3H]thymidine triphosphate incorporation into isolated nuclei. After PKC induction, ADR activity is increased (890 +/- 120 vs. 3910 +/- 345 cpm, P < 0.001). In the presence of the PKC inhibitor H7, ADR activity fails to increase (100 +/- 50 vs. 3910 +/- 345 cpm, P < 0.001). The high levels of ADR found in PHA-stimulated cells is marginally affected by in situ PKC induction, although the sustained impact of H7 throughout the cell cycle suggests that ADR is constantly being phosphorylated. Titration of CsA or RAPA into the cell-free system inhibited ADR induction in a dose-dependent fashion. The utility of ADR induction as a marker for immunosuppression was investigated by comparing ADR inducibility in resting cells that had been preloaded with CsA or RAPA with the proliferative index of cells cultivated with CsA or RAPA. ADR induction directly correlated with CsA or RAPA proliferative inhibition. The results suggested that ADR is a constituent of the PKC phosphoregulatory cascade essential for cell cycle progression. The correlation between cell-free ADR inducibility and proliferative inhibition by CsA or RAPA suggest that this procedure may be useful for in vitro prediction of allografted patient immunologic response.