REV5901, an antagonist of cysteinyl-leukotriene receptors and an inihibitor of 5-lipoxygenase (1, 2), has been recently shown to inhibit regulatory volume decrease (RVD) in articular chondrocytes (3). Extracellular matrix (ECM) synthesis has been shown to be dependent upon extracellular osmolality and thus cell volume whereby chondrocytes have the capacity to resist changes in cell volume via RVD. Currently, the mechanism of RVD is unknown and therefore as REV5901 inhibits the volume regulatory response here we have investigated the effects of REV5901 loading on chondrocyte intracellular calcium as a potential signalling pathway. Bovine articular cartilage explants were excised from load-bearing surfaces of the metacarpal-phalangeal joints of animals (~30mths) on the day of slaughter from the local abattoir and chondrocytes isolated using a standard technique (4). Changes in intracellular calcium were determined using calcium indicator Fluo4 (5μM; [5]) and a fluorescent plate reader and data expressed as mean ± standard error of the mean. Student’s T-Tests were performed and values deemed significant for P<0.05. N=4 animals from 5 distinct experiments. There was significant (P<0.05; Students T-Test) and sustained rise in [Ca2+]i in response to REV5901 loading reaching 33.78±5.66% after a 5min incubation period. This rise was reduced to 27.9±3.2% following the chelation of extracellular calcium with 2mM EGTA suggesting the contribution of an intracellular calcium store to the REV5901-induced [Ca2+]i rise. Both the PLCβ3 and PI5K pathways were therefore studied using individual inhibitors against key signal transduction molecules. In the presence of a PLCβ3 inhibitor U73122 (100μM) or an inhibitor of IP3 formation Neomycin (10mM) there was a significant decrease in maximal calcium rise to 23.09±2.70% or 23.78±2.21% (P<0.05; Student’s T-Test), respectively, indicating the involvement of both PLCβ3 and PIP2 molecules in mediating the effects of REV5901. The use of Neomycin also demonstrated a delay in the calcium rise by ∼28.8 seconds possibly due to the overproduction of PIP2 molecules, whereas PLCβ3 inhibition reduced the rate of calcium rise from 3.82±0.42%/min to 1.75±0.19%/min. To confirm the involvement of the PLCβ3 pathway, inhibition with PKC inhibitor Rottlerin (100μM) abolished the REV5901-induced [Ca2+]i rise. Finally the use of PI5K inhibitor Wartmannin (1μM) did not have a significant effect on calcium rise. These data show a PLCβ3 and PKC-dependant release of calcium from intracellular stores in response to REV5901 loading, thus suggesting a mode of action for the inhibitory effects of REV5901 on chondrocyte RVD.