The objective was to determine treatment dose and time-dependent effects of injected mitomycin C on extracellular matrix fibroblasts, collagen, and fibronectin, important mediators in the wound healing response, in a rat cutaneous wound model. A prospective, controlled animal study. Forty rats were injected with three different doses (0.4, 2.3, and 5.0 mg/mL) of mitomycin C at three different wound sites with a fourth wound site receiving saline as a control. The rats were grouped to have their tissue harvested at five different dates ranging from 1 week to 8 weeks. After death, samples from the wound site underwent Western blot analysis for collagen and fibronectin and histological analysis measuring fibroblast apoptosis. Over an 8-week period, collagen and fibronectin significantly decreased and fibroblast apoptosis significantly increased. No correlation was found between the injected dose of mitomycin C and either the extracellular matrix protein concentration or the rate of fibroblast apoptosis. Mitomycin C demonstrated a long-term effect in a wound, inhibiting collagen and fibronectin production and inducing apoptosis. Use of mitomycin C in excess of 0.4 mg/mL did not alter protein concentrations or rate of apoptosis.