Hypothesis: Fibroblasts, not myofibroblasts, are responsible for wound contraction. Only myofibroblasts express a smooth muscle actin for which vanadate blocks its expression. Wound contraction in vanadate-treated rats will proceed normally in the absence of myofibroblasts. Design: Laboratory study using rats. Methods: Wound healing in rats receiving vanadate parenterally, an inhibitor of tyrosine phosphate phosphatases, was investigated. For 21 days, treated rats received drinking water containing vanadate, 0.2 mg/mL, in isotonic sodium chloride solution, and the control rats received isotonic sodium chloride solution alone. On day 7, 4 square, full-excision wounds were made dorsally and measured, then 2 polyvinyl alcohol sponges were placed ventrally in subcutaneous pockets. Results: After 2 weeks, the wound area in the rats receiving vanadate measured 7.1+/-1.8 U (mean+/-SD), and the wound area in the control rats measured 7.2 +/- 2.2 U. The control rats' granulation tissue (GT) had myofibroblasts, or alpha-smooth muscle (alpha-SM) actin-positive fibroblasts, whereas the vanadate-treated group's fibroblasts were devoid of alpha-SM actin. By Western blot analysis, GT homogenates in the vanadate-treated group contained less alpha-SM actin. By electron microscopy, control rats' GT showed classic myofibroblast populations, and the collagen fiber bundles were randomly organized. In contrast, the wounds in the vanadate-treated group showed unencumbered fibroblast populations and neatly ordered, parallel collagen fiber bundles. By polarized light microscopy, the GT of the vanadate-treated group displayed orderly collagen fiber bundles. Conclusions: The differentiation of fibroblasts into myofibroblasts requires the dephosphorylation of selected tyrosine phosphate residues. In the absence of myofibroblasts, the rate of rat wound contraction is normal, and collagen fiber bundles have a more orderly arrangement. Myofibroblasts are not required for wound contraction.