• We have studied the modulation of volume-regulated anion channels (VRACs) by the small GTPase Rho and by one of its targets, Rho kinase, in calf pulmonary artery endothelial (CPAE) cells. • RT-PCR and immunoblot analysis showed that both RhoA and Rho kinase are expressed in CPAE cells. • ICl,swell, the chloride current through VRACs, was activated by challenging CPAE cells with a 25 % hypotonic extracellular solution (HTS) or by intracellular perfusion with a pipette solution containing 100 M GTPS. • Pretreatment of CPAE cells with the Clostridium C2IN-C3 fusion toxin, which inactivates Rho by ADP ribosylation, significantly impaired the activation of ICl,swell in response to the HTS. The current density at +100 mV was 49 ± 13 pA pF−1 ( n= 17) in pretreated cells compared with 172 ± 17 pA pF−1 ( n= 21) in control cells. • The volume-independent activation of ICl,swell by intracellular perfusion with GTPS was also impaired in C2IN-C3-pretreated cells (31 ± 7 pA pF−1, n= 11) compared with non-treated cells (132 ± 21 pA pF−1, n= 15 ). • Activation of ICl,swell was pertussis toxin (PTX) insensitive. • Y-27632, a blocker of Rho kinase, inhibited ICl,swell and delayed its activation. • Inhibition of Rho and of Rho kinase by the above-described treatments did not affect the extent of cell swelling in response to HTS. • These experiments provide strong evidence that the Rho-Rho kinase pathway is involved in the VRAC activation cascade.