Changes in microtubule polymerization state have been shown to affect many cellular events, including the contractile properties of smooth muscle. We have previously shown that depolymerization of microtubules causes significant vasoconstriction in arterioles. This vasoconstriction does not require the endothelium or an increase in vascular smooth muscle Ca<sup>2+</sup>. Consequently, we hypothesized that a Ca<sup>2+</sup>-sensitizing mechanism may be involved in the constrictor response. The purpose of these experiments was to further elucidate cell signaling pathways responsible for vasoconstriction following microtubule disruption. Rat skeletal muscle arterioles were isolated, cannulated and pressurized without intraluminal flow. All arterioles used for experiments developed spontaneous, myogenic tone (54% of passive diameter). Microtubule depolymerization with colcemid or vinblastine caused arterioles to constrict by an additional 20% from resting basal diameter. In addition, arterioles treated with colcemid showed significantly enhanced responsiveness to norepinephrine and reduced responsiveness to adenosine. To investigate a role for Rho-kinase, vessels were incubated with inhibitors of the Rho-kinase pathway – Y-27632 or C3 exoenzyme. Inhibition of Rho-kinase significantly inhibited the constriction associated with colcemid-induced microtubule depolymerization. Inhibition of Rho-kinase also abolished the increased responsiveness to norepinephrine whereas adenosine responsiveness continued to be reduced. By comparison, inhibition of the tyrosine kinase, Src, with PP2 did not have any effect on the colcemid-induced changes in vascular tone or reactivity. These data indicate that the vasoconstriction and enhanced norepinephrine reactivity associated with microtubule disruption involves a Ca<sup>2+</sup>-sensitization process that is mediated by the Rho-kinase pathway.