We investigated the mechanisms underlying regulation of contraction with measurements of isometric force and intracellular calcium concentration ([Ca²⁺]_i) in NIH 3T3 fibroblast re-constituted into fibers using a collagen matrix. Treatment with the major phospholipids, neurotransmitters and growth factors had little effect on baseline isometric force. However, U46619, a thromboxane A₂ (TXA₂) analogue, increased force and [Ca²⁺]_i; EC₅₀ values were 11.0 and 10.0 nM, respectively. The time courses were similar to those induced by calf serum (CS) and the maximal force was 65% of a CS-mediated contraction. The selective TXA₂ receptor antagonist, SQ29548, abolished the U46619-induced responses. CS-induced contractions are dependent on an intracellular calcium store function; however, the U46619 response depended not only on intracellular calcium stores but also calcium influx from the extracellular medium. Inhibition of Rho kinase suppressed both U46619- and CS-induced responses; in contrast, inhibition of C kinase (PKC) reduced only the U46619 response. Moreover, addition of U46619 to a CS contracture enhanced force and [Ca²⁺]_i responses. These results indicate that U46619-induced responses involve both PKC and Rho kinase pathways, in contrast to activation by CS. TXA₂ thus may have a role in not only the initial step of wound repair as an activator of blood coagulation, but also in fibroblast contractility in later stages.