Pulmonary irradiation fibrosis involves migration to the lungs of bone marrow origin myofibroblast progenitor cells (marrow stromal cells (MSCs)). Smad3-/- mice display decreased ionizing irradiation-induced skin fibrosis, defective osteochondrogenesis and other abnormalities thought to be associated with a defective stromal cell response(s) to transforming growth factor-beta (TGFFbeta). Clonal bone marrow stromal cell lines were derived from the adherent layer of continuous bone marrow cultures of homozygous deletion recombinant negative Smad3-/- mice and Smad3+/+ littermates. Quantitation in an Automated Cell Tracking System of the in vitro single cell migratory capacity over five days demonstrated a significant decrease in locomotion in microns per 24 h of Smad3-/- compared to Smad3+/+ clonal MSC lines. Reexpression by retroviral vector transfection of the Smad3 but not control ds-red transgene restored in vitro migratory capacity. Intravenously injected GFP transgene product labeled Smad3-/- (MSCs) seeded 10-fold less effectively than ds-red transgene product labeled Smad3+/+ cells to the 80 days post 20 Gy irradiated lungs of C57BL/6J mice and proliferated less significantly for 60 days after cell injection. Female mice chimeric for male Smad3-/- compared to Smad3+/+ marrow showed decreased irradiation pulmonary fibrosis, Y+ stromal cell migration to the lungs, and improved survival. The data show that the reduced in vitro and in vivo migratory capacity of Smad3-/- bone marrow stromal cells correlates with decreased radiation pulmonary fibrosis observed in mice chimeric for Smad3-/- marrow.