Drosophila melanogaster possesses a single gene, Dm myb, that is closely related to the vertebrate family of Myb genes, which encode transcription factors that are involved in regulatory decisions affecting cell proliferation, differentiation and apoptosis. The vertebrate Myb genes have been specifically implicated in regulating the G(1)/S transition of the cell cycle. Dm myb is expressed in all proliferating tissues, but not at detectable levels in endoreduplicating cells. Analysis of loss-of-function mutations in Dm myb revealed a block at the G(2)/M transition and mitotic defects, but did not directly implicate Dm myb function in the G(1/)S transition. We have used the Gal4-UAS binary system of ectopic expression to further investigate the function of Dm myb. Our results demonstrate that depending upon the type of cell cycle, ectopic Dm myb activity can exert opposing effects on S phase: driving DNA replication and promoting proliferation in diploid cells, even when developmental signals normally dictate cell cycle arrest; but suppressing endoreduplication in endocycling cells, an effect that can be overcome by induction of E2F. We also show that a C-terminally truncated DMyb protein, which is similar to an oncogenic form of vertebrate Myb, has more potent effects than the full-length protein, especially in endoreduplicating tissues. This finding indicates that the C terminus acts as a negative regulatory domain, which can be differentially regulated in a tissue-specific manner. Our studies help to resolve previous discrepancies regarding myb gene function in Drosophila and vertebrates. We conclude that in proliferating cells, Dm myb has the dual function of promoting S phase and M phase, while preserving diploidy by suppressing endoreduplication.