The IMP dehydrogenase inhibitors mycophenolic acid (MPA) and tiazofurin (TZ) induce a time- and dose-dependent inhibition of cell growth, as well as differentiation in T-lymphoid CEM-2 leukemia cells. The differentiated cells have acquired a suppressor/cytotoxic T-lymphocyte phenotype characterized by reactivity with maturation-specific monoclonal antibodies. Coadministration of guanosine and hypoxanthine reduces the growth inhibition and diminishes the induction of differentiation by either MPA or TZ. No such reduction was observed for differentiation induced by phorbol 12-myristate 13-acetate (PMA), another inducer of a suppressor/cytotoxic phenotype in CEM-2 cells. During the first 2 days of treatment with MPA or TZ, a pattern of stable IMPDH mRNA levels and increased amounts of cellular enzyme was observed, perhaps, because of compensation for the inhibitor-mediated decrease in cellular IMPDH activity or a MPA- or TZ-mediated decrease in proteolysis of IMPDH. PMA treatment decreased the levels of IMPDH mRNA, protein, and activity. In addition, treatment of CEM-2 cells with either IMPDH inhibitors or PMA caused different alterations of the ribonucleotide pools. The lack of a consistent pattern of IMPDH expression in CEM-2 cells treated with IMPDH inhibitors or PMA indicates that no general association exists between the induction of cell differentiation and the expression of IMPDH. Nevertheless, our results indicating that IMPDH inhibitors can induce differentiation in CEM-2 cells suggest that this treatment may provide a useful approach to circumvent the differentiation block in some tumor cells.