Rho kinase is an enzyme that has been implicated in pathological wound healing. In our study, we aimed to characterize the dosages of Y-27632 (a competitive inhibitor of rho kinase) at which fibroblast stress fiber formation is inhibited, to determine the effect of Y-27632 on fibroblast confluence, and to determine the effect of varying concentrations of Y-27632 on fibroblast-mediated gel contraction. First, 3T3 fibroblasts were exposed to various concentrations of Y-27632, and stress fiber dissolution was assessed with immunofluorescence. Next, 10 microM Y-27632 was applied to 3T3 fibroblast monolayer cultures immediately after plating and at 8 hours after plating to determine the effect on fibroblast confluence. Finally, a three-dimensional model was used to assess the effect of Y-27632 on gel contraction. Increasing doses of Y-27632 resulted in a dose-dependent increase in stress fiber dissolution as well as a dose-dependent inhibition of gel contraction. Median number of days to confluence were 3, 5, and 6 after no, immediate, and delayed incubation with Y-27632, respectively (P < .05). Our results further characterize the dosages at which Y-27632 may be most effective in the modulation of wound contraction in burn patients. In addition, our confluence study suggests that patients need not necessarily be pretreated with Y-27632.