The c-myb proto-oncogene product (c-Myb) is a transcriptional activator. Vertebrate c-Myb is a key regulator of the G(1)/S transition in cell cycle, while Drosophila Myb (dMyb) is important for the G(2)/M transition. Here we report that dMyb induces expression of cyclin B, a critical regulator of the G(2)/M transition, in Drosophila eye imaginal disc. In the wild-type eye disc, dmyb mRNA was expressed in the stripes both anterior and posterior to the morphogenetic furrow. Ectopic expression of C-terminal-truncated dMyb in the eye disc caused ectopic expression of cyclin B and the rough eye phenotype. This rough eye phenotype correlated with prolonged M phase, caused by overexpression of cyclin B. Cyclin B expression was lost in dmyb-deficient clones. In Schneider cells, the activity of the cyclin B promoter was dramatically reduced by loss of dMyb using the RNA interference method. Mutations of the multiple AACNG sequences in the cyclin B promoter also abolished the promoter activity. These results indicate that dMyb regulates the G(2)/M transition by inducing cyclin B expression via binding to its promoter.