P2Y receptors are a family of G protein-coupled receptors that are activated by endogenous nucleotides, such as ATP, UTP and UDP. In the vascular system these agonists induce vasodilation via endothelial P2Y receptors and vasoconstriction via P2Y receptors located on arterial smooth muscle cells (Chootip et al., 2002). The intracellular signalling mechanisms by which vasoconstriction is induced is poorly characterised, so the aim of this study was to determine the role of Ca²⁺-sensitisation of the contractile proteins in nucleotide-evoked vasoconstriction of rat small intrapulmonary artery (SPA). 5 mm rings of rat SPA, from which the endothelium was removed by gentle rubbing of the intima, were mounted under isometric conditions in 1ml baths at 37°C and a resting tension of 0.5g. Tension was recorded by Grass FT 03 transducers connected to a Powerlab/4e system (AD Instruments). Contractions were elicited by addition of agonists to the bath. UTP and UDP (both 300 μM) evoked slowly developing contractions, which reached a peak within 5 minutes and decayed slowly in the continued presence of agonist. The contractions were of similar amplitude when UTP and UDP were administered at 30 minute intervals. Both protein kinase C (PKC) and rho kinase have been implicated in Ca²⁺-sensitisation of smooth muscle, therefore we investigated the effects of selective inhibitors of these enzymes. The PKC inhibitor GF 109203X (3 μM) abolished contractions evoked by the PKC activator phorbol 12-myristate13-acetate (PMA) (10 μM) when applied beforehand and reversed the contractions if applied once they had developed. In contrast, it had only a small effect on the UTP and UDP-evoked responses. The rho kinase inhibitor Y27632 (10 μM) had no effect on PMA-induced responses, consistent with a selective action against rho kinase, but caused a moderate inhibition of nucleotide-evoked contractions. These results indicate that Ca²⁺-sensitisation is involved in P2Y receptor-mediated contraction of rat pulmonary artery. Furthermore, rho kinase plays a greater role than PKC. Other signalling mechanisms, most likely Ca²⁺ influx and release, also appear to be involved and make a greater contribution.