Aurora-A kinase (AurA) is a key regulator of cellular processes involving microtubules. It has also been implicated in actin-dependent events, but the mechanisms that underlie the processes are not fully understood. Here we provide genetic and biochemical evidence suggesting that AurA negatively regulates Drok, the only known Rho-kinase orthologue in Drosophila. AurA directly phosphorylates Drok in vitro, and the overexpression of the nonphosphorylatable forms of Drok in vivo causes similar, but much stronger effects than that of wild-type Drok. The defects induced by the nonphosphorylatable forms of Drok are compensated by reducing the function of myosin downstream. Thus, phosphorylation of Drok by AurA normally suppresses Drok activity. We propose that AurA directly regulates actin-dependent processes by phosphorylating Rho-kinase. (C) 2013 The Authors. Published by Elsevier Inc. All rights reserved.