OBJECTIVES This study aims to investigate the effect of mitomycin-C in the wound healing process on collagen synthesis in tracheostomyzed rats. MATERIALS AND METHODS Fourteen healthy, in both sexes, mean weight of 270 g (range 250-300 g), Wistar-Albino type rats underwent tracheotomy and tracheal mucosa was damaged with micro-scissors on both sides of tracheostomyzed area. The rats were divided into two groups: The experimental group (group 1) received immediate topical application of mitomycin-C 0.2 mg/ml; the control group (group 2) received saline solution. The rats were sacrificed after a period of one month. Subsequently, the tracheostomyzed region was excised and vertically divided into the two parts. The level of hydroxyproline in the dry tissue was measured in one part of the tissue. Fibroblast count was performed in the other part of the tracheostomyzed region using the stereological method. RESULTS The hydroxyproline level was much higher in the mitomycin-C group when compared to the control group (p<0.05). In contrary, the number of fibroblasts was lower in the mitomycin-C group than control group (p<0.05). CONCLUSION When used in wound healing, mitomycin-C may increase collagen synthesis or quicken the wound healing process after one month.