Airway smooth muscle (ASM) mass appears to be increased in the bronchi of patients with chronic severe asthma. Although the precise mechanisms that induce these changes are unknown, increases in ASM mass are caused, in part, by ASM cell proliferation. After allergen challenge in rats, it has been possible to demonstrate an increase in ASM mass by morphometric techniques. To examine whether hyperplasia is involved in ASM cell growth in vivo, we investigated whether repeated allergen challenges in sensitized Brown Norway rats stimulated DNA synthesis in airway epithelial and ASM cells. Animals that were actively sensitized to ovalbumin (OA) received either three aerosolized OA or saline challenges at 5-day intervals. DNA synthesis was measured by indirect immunohistochemical techniques with an anti-bromodeoxyuridine (BrdU) antibody. OA inhalations increased ASM mass as determined by morphometry and also induced DNA synthesis in both airway epithelial and ASM cells in the airways of sensitized animals compared with saline-challenged control animals. ASM mass was increased in large- and medium-sized airways but not in small airways. However, the number of BrdU-positive ASM cells normalized to basement membrane length was also greater in the large- and medium-sized airways compared with that in the small airways. When the number of BrdU-positive epithelial cells was normalized to basement membrane length, there was no difference among airway sizes and the number of BrdU-positive epithelial cells. These data suggest that DNA synthesis is induced in both airway epithelial and ASM cells after inhalational antigen challenge.