Binucleine 2 is an isoform-specific and ATP-competitive inhibitor of Drosophila Aurora B kinase (Ki = 0.36 µM), a kinase involved in cell division. It is specific for Drosophila Aurora B kinase, inhibiting it in a dose-dependent manner, with minimal inhibition of human or X. laevis Aurora B kinases at concentrations up to 100 µM. Binucleine 2 induces mitotic and cytokinesis defects in Drosophila Kc167 cells. It prevents Drosophila S2 cells from assembling a contractile ring during cell division when used at a concentration of 40 µM but does not affect ring ingression, suggesting that Aurora B kinase activity is not required for that step.