A specific calmodulin-binding protein of 68 kDa (CaM-BP68) is modulated in response to growth factors that induce proliferative stimulation in a variety of hemopoietic progenitor cells. The nuclear localization of the CaM-BP68 coincided temporally with interleukin 3 (IL-3)-dependent progression of synchronized FDC-P1 cells from G1 to S phase [Reddy et al. (1992) Blood 79, 1946-1956]. To delineate the role of the CaM-BP68 in the onset of DNA synthesis (S phase), this protein was purified to an apparent homogeneity from FDC-P1 cells and its effects on DNA replication in permeabilized FDC-P1 cells were examined. Purified CaM-BP exhibited a single silver-stained protein band of 68 kDa on SDS-polyacrylamide gels. This purified protein, when incubated with permeabilized log-growing FDC-P1 cells, caused a 3-4-fold increase in the rate of [3H]dTTP incorporation into DNA as compared to the controls. There was a direct correlation between the increase in the rate of [3H]dTTP incorporation into DNA and the concentration of the added CaM-BP68 in the incubation mixture. These observations suggest that the CaM-BP68, whose nuclear localization is associated with growth factor dependent proliferative stimulation of myeloid progenitor cells, is involved in the regulation of nuclear DNA synthesis.