Our objectives were to identify the relative contributions of intracellular Ca²⁺ concentration ([Ca²⁺]_i) and myofilament Ca²⁺ sensitivity in the pulmonary venous smooth muscle (PVSM) contractile response to the thromboxane A₂ mimetic, U46619, and to assess the roles of protein kinase C (PKC), tyrosine kinases (TK) and rho-kinase (ROK) in that response. We tested the hypothesis that U46619-induced contraction in PVSM is mediated both by increases in [Ca²⁺]_i and myofilament Ca²⁺ sensitivity, and that the PKC, TK and ROK signaling pathways are involved. Isometric tension was measured in isolated endothelium-denuded (E-) canine pulmonary venous (PV) rings. In addition, [Ca²⁺]_i and tension were simultaneously measured in fura-2 loaded E- PVSM strips. U46619 (0.0001-1 µM) caused dose-dependent (P<0.001) contraction in PV rings. U46619 contraction was attenuated by inhibitors of L-type voltage-operated Ca²⁺ channels (nifedipine) (P<0.001), IP₃-mediated Ca²⁺ release (2-APB) (P<0.001), PKC (bisindolylmaleimide I) (P<0.001), TK (tyrphostin A 47) (P=0.014) and ROK (Y27632) (P=0.008). In PV strips, U46619 contraction was associated with increases in [Ca²⁺]_i and myofilament Ca²⁺ sensitivity. Both Ca²⁺ influx and release mediated the early transient increase in [Ca²⁺]_i, whereas the late sustained increase in [Ca²⁺]_i only involved Ca²⁺ influx. Inhibition of both PKC and ROK (P=0.006 and P=0.002, respectively), but not TK, attenuated the U46619-induced increase in myofilament Ca²⁺ sensitivity. These results suggest that U46619 contraction is mediated by Ca²⁺ influx, Ca²⁺ release and an increase in myofilament Ca²⁺ sensitivity. The PKC, TK and ROK signaling pathways are involved in U46619 contraction.