We investigated cellular mechanisms that either mediate or modulate the vascular response to muscarinic receptor activation (acetylcholine (ACh)) in pulmonary veins (PV). Isometric tension was measured in isolated canine PV rings with endothelium (E+) and without endothelium (E-). Tension and intracellular Ca²⁺ concentration ([Ca²⁺]_i) were measured simultaneously in fura-2 loaded E- PV strips. In the absence of preconstriction, ACh (0.01-10 µM) caused dose-dependent (P<0.001) contraction in E+ and E- rings. ACh contraction was potentiated by removing the endothelium (P<0.001) or by nitric oxide (NO) synthase inhibition (N-nitro-L-arginine methylester) (P=0.001). Cyclooxygenase inhibition (indomethacin) reduced ACh contraction in both E+ and E- PV rings (P=0.013 and P=0.037, respectively). ACh contraction was attenuated by inhibitors of voltage-operated Ca²⁺ channels (nifedipine) (P<0.001), IP₃-mediated Ca²⁺ release (2-APB) (P<0.001), protein kinase C (bisindolylmaleimide I) (P<0.001), rhokinase (Y27632) (P=0.02) and tyrosine kinase (tyrphostin 47) (P=0.015) in E- PV rings. ACh (1 µM) caused a leftward shift in the [Ca²⁺]_i-tension relationship (P=0.015); i.e. ACh increased myofilament Ca²⁺ sensitivity. Inhibition of protein kinase C, rho-kinase and tyrosine kinase attenuated the ACh-induced increase in myofilament Ca²⁺ sensitivity (P<0.001, P<0.001 and P=0.024, respectively). These findings indicate that in canine PV, ACh contraction is modulated by NO and partially mediated by metabolites of the cyclooxygenase pathway, and involves Ca²⁺ influx through voltage-operated Ca²⁺ channels and IP₃-mediated Ca²⁺ release. In addition, ACh induces an increase in myofilament Ca²⁺ sensitivity which requires the protein kinase C, rho-kinase and tyrosine kinase pathways.