CBFβ-SMMHC is expressed from the inv (16) chromosome in M4Eo AML. Mice lacking CBF subunits or expressing the CBFβ-SMMHC or AML1-ETO oncoproteins failed to develop definitive hematopoiesis. To investigate these effects on hematopoiesis, we expressed CBFβ-SMMHC from the metallothionein promoter, in both 32D cl3 myeloid cells and Ba/F3 B-lymphoid cells. Addition of zinc increased CBFβ-SMMHC levels more than tenfold, with higher levels evident in Ba/F3 lines. Levels obtained in 32D cl3 cells were similar to those of endogenous CBFβ. Indirect immunofluorescence revealed zinc-inducible speckled, nuclear staining in Ba/F3 cells and diffuse nuclear staining in 32D cl3 cells. CBFβ-SMMHC reduced endogenous CBF DNA-binding fivefold in both cell types, increased cell generation time 1.9-fold, on average, in 32D cl3 cells and 1.5-fold in Ba/F3 cells and decreased tritiated thymidine incorporation into DNA correspondingly. CBFβ-SMMHC increased the proportion of cells in G1 1.7-fold, on average, in 32D cl3 and Ba/F3 cells, and decreased the proportion of cells in S phase by a similar degree. CBFβ-SMMHC induced a marked increase in hypophosphorylated Rb, but did not alter IL-3 Receptor α or β subunit levels. Neither apoptosis nor 32D differentiation was induced by zinc in IL-3 in these lines. Induction of CBFβ-SMMHC in 32D cl3 cells did not inhibit their differentiation to neutrophils or their expression of myeloperoxidase mRNA in G-CSF, and did not produce an eosinophilic phenotype. Additional, proliferative genetic changes in M4eo AMLs might potentiate inhibition of differentiation by CBFβ-SMMHC by allowing its increased expression.