We investigated the effects of Bordetella bronchiseptica dermonecrotic toxin on DNA synthesis in MC3T3-E1 cells. The rate of [methyl-3H]thymidine incorporation increased in the toxin-treated cells more than 24 h after addition of the toxin under the serum-starved conditions. This effect was dependent on the toxin concentration ranging from 0.3 to 3 ng/ml and was eliminated by aphidicolin and hydroxyurea, inhibitors for DNA replication. In the toxin-treated culture, the number of cells did not increase but polynucleated cells appeared and their number increased to ca. 50% of the total number of cells 6 days after the toxin addition. From these results, we concluded that the toxin stimulates DNA replication in MC3T3-E1 cells without cell proliferation.