Protease-activated receptor 2 (PAR2) is widely distributed throughout the central nervous system (CNS) and has been shown to play a role in neurogenic inflammation and nociception (MacFarlane et al, 2001). In hippocampal cultures, chronic activation of PAR2 leads to cell death and is associated with elevated [Ca²⁺]_i (Smith-Swintosky et al, 1997) but the signalling mechanisms underlying this remain unclear. In the present study, we have examined the pathways underlying PAR2 [Ca²⁺]_i responses in cultured hippocampal neurones, using fluorescence-imaging techniques.Cultures of rat hippocampal neurones were prepared from humanely killed rats and [Ca²⁺]_i measured as described previously (Shanley et al, 2002). Experiments were performed on cells >13 days in culture, continually perfused with a standard HEPES-buffered saline containing TTX (0.5µmM). All drugs were applied via the perfusate. Data are expressed as mean ± S.E.M. of fluorescent ratio arbitrary units (a.u.) and statistical significance determined using Student’s paired t-tests, n = number of neurones obtained from at least two separate cultures.In the majority of neurones, application of trypsin (80nM) elevated [Ca²⁺]_i from control levels by 0.35 ± 0.05 a.u. (n=17/20, P<0.01). In addition, the selective PAR2 peptide agonists, SLIGRL (80µM) and ASKH95 (80µM), both elevated [Ca²⁺]_i from control levels by 0.15 ± 0.02 a.u. (n=26, P<0.01) and 0.23 ± 0.03 a.u. (n=15, P<0.01) respectively. In Ca²⁺-free solution, SLIGRL (80µM) increased [Ca²⁺]_i,however responses were moderately reduced (20 ± 5 % reduction, n=10, P<0.01). Application of SLIGRL following the depletion of intracellular Ca²⁺ stores with cyclopiazonic acid (30µM) had little effect on [Ca²⁺]_i (97 ± 4% reduction, n=10, P<0.01) indicating intracellular stores as the primary source of the elevated [Ca²⁺]_i.Inhibition of PLC and IP3 receptors by U73122 (1µM) and 2-aminoethoxydiphenyl borate (20µM) also reduced SLIGRL responses (87 ± 4% reduction, n=10, P<0.01 and 91 ± 5% reduction, n=10, P<0.01, respectively).These data reveal that activation of PAR2 in neurones elevates [Ca²⁺]_i,via the PLC / IP3 pathway. Thus PAR2 may play an important role in modulating Ca²⁺-dependent processes within the hippocampus.