Articular chondrocytes are exposed to significant changes in extracellular osmolarity during normal joint activity, which can lead to changes in cell volume and metabolism of the extracellular matrix (ECM). Chondrocytes can respond to cell swelling/shrinking by volume regulatory pathways, but the signalling pathways are poorly understood although a role for the cytoskeleton is frequently implicated. Here, we have investigated the effects of disruption of the chondrocyte F-actin cytoskeleton on the recovery of cell volume by RVD. The cytoskeleton was perturbed using the relatively specific agent latrunculin B (5 μM; 30 min) and loss of F-actin integrity quantified using fluorescent phalloidin-labelling and confocal laser scanning microscopy (CLSM). Imaging of isolated chondrocytes labelled with Fura-2 to measure the fluorescence associated with cell volume changes, showed that the extent of hypo-osmotic swelling was unaffected by latrunculin B treatment. Two categories of the chondrocyte RVD response were observed: 'fast' RVD where at 3 min post-osmotic challenge there was a recovery in cell fluorescence of ≥80%, whereas other cells exhibited 'slow' RVD. Latrunculin B increased the proportion of chondrocytes demonstrating 'fast' RVD by ∼10 fold and reduced those cells showing 'slow' RVD. An inhibitor of chondrocyte RVD (REV 5901) had no significant effect on the integrity of the cytoskeleton showing that the RVD response could be inhibited independent of the state of the F-actin cytoskeleton. These results suggest that the intact cortical F-actin cytoskeleton has a restraining effect on the RVD response of isolated bovine articular chondrocytes.