Alveolar epithelial injury occurs universally in common respiratory illnesses associated with diffuse lung damage. After alveolar injury, type II cells proliferate and reestablish epithelial integrity, thereby restoring normal lung structure and function. However, the regulation of type II cell proliferation and alveolar epithelial repair is poorly understood. Hepatocyte growth factor/scatter factor (HGF/SF) is a heparin-binding growth factor that has been shown to be mitogenic for cultured alveolar type II cells. In this study, we determined the effect of intratracheal instillation of rhHGF/SF on type II cell proliferation in vivo. To quantify the alveolar type II cell proliferative response, we developed a double-label immunohistochemical technique to detect replicating alveolar type II cells in formalin-fixed lung sections that utilized the identification of proliferating cells by bromodeoxyuridine (BrdUrd) incorporation into DNA and alveolar type II cells by 3F9 immunoreactivity. BrdUrd detection was optimized by enzymatic antigen recovery and silver intensification of the horseradish peroxidase reaction product. Intratracheal instillation of rhHGF/SF induced a time- and dose-dependent increase in type II cell proliferation. The type II cell labeling index increased to 12.3 +/- 6.0% 48 h after 1.0 mg/kg rhHGF/SF administration, compared with 2.6 +/- 0.9% after PBS instillation. To compare the normal type II cell reparative response with the level of proliferation after exogenous rhHGF/SF administration, we measured the specific alveolar type II cell labeling index in rat lung sections obtained from animals exposed to hyperoxia for 50 h and then allowed to recover in room air. After 1 day of recovery, the alveolar type II cell labeling index was 0.45 +/- 0.2%. The specific labeling index increased to 5.4 +/- 1.3% at 2 days and then declined to 0.31 +/- 0.16% 5 days after hyperoxia exposure. In animals not exposed to hyperoxia, the alveolar type II cell labeling index was 0.6 +/- 0.14%. These studies demonstrated that intratracheal instillation of rhHGF/SF promoted alveolar type II cell proliferation in vivo. The maximal level of type II cell proliferation after rhHGF/SF administration was more than twice that reached during recovery from hyperoxia exposure. Thus, intratracheal instillation of HGF/SF may provide a potential strategy to promote type II cell proliferation and augment alveolar epithelial repair after lung injury.