Agents that alter the morphology of rabbit synovial fibroblasts induce synthesis of the metalloendopeptidases, collagenase and stromelysin. We studied the relationship of cytoskeletal changes to the commitment to expression of these metalloendopeptidases. Cells treated with cytochalasin B (CB) or 12-O-tetradecanoylphorbol-13-acetate rounded, and only cells that had lost their stress fibers expressed collagenase and stromelysin, as determined by immunofluorescence. We concentrated on the effects of CB because of its rapid reversibility. When CB was added for 1-24 h, then removed, the cells respread within 30-60 min. The minimum period of CB treatment that committed cells to the subsequent synthesis of collagenase and stromelysin was 3 h. After initial treatment with 2 micrograms/ml CB for 3-24 h, or with various concentrations of CB (0-2 micrograms/ml) for 24 h, both enzyme activity and biosynthesis of the proenzymes showed a graded increase when measured at 24 h. Even after treatment with 2 micrograms/ml CB for only 3 h, greater than 85% of all cells were positive for both collagenase and stromelysin when cells were monitored by immunofluorescence. In contrast, when the dependence of collagenase and stromelysin expression on the inducing concentration of CB was examined, there was a dose-dependent increase in the number of cells positive for collagenase and stromelysin, as determined by immunofluorescence. Thus, at low concentrations of CB (less than 0.5 micrograms/ml), a heterogeneous population response was observed. These results suggest that the commitment of fibroblasts to induction of the metalloproteinases is a stochastic process in which a second signal that correlates with the disruption of the actin cytoskeleton may be rate-limiting for collagenase and stromelysin gene expression.