Little is known of the molecular basis of smooth-muscle cell development in the microvessels of the adult lung in pulmonary hypertension (PH). Using quantitative and immunogold electron microscopy techniques we report the development of microvascular precursor smooth-muscle cells (PSMCs) expressing α-smooth-muscle actin (αSMA), a first marker of smooth-muscle cell differentiation, in rats with hyperoxic PH. Increase in the frequency of distal (alveolar wall) vessels with αSMA cells preceded (P _χ² < 0.02, Day 4) the increase in proximal (alveolar duct) vessels (P _χ² < 0.02, Day 14). The smallest vessel with cells expressing αSMA (< 50 μm in diameter) increased most with time (P _χ² < 0.001). Immunopositive PSMCs were rare in normal lung and frequent in hyperoxia. Well-developed filament arrays decorated with αSMA were detected in intermediate cells early in hyperoxia (Day 4). Similar filament networks were detected later in fibroblasts recruited to vessel walls (Days 7 to 14). By Day 28, cells derived from fibroblasts formed several layers in the vessel wall and expressed dense αSMA filament arrays, in either a central domain or mesh. Thus, intermediate cells are the source of cells expressing αSMA early in the microvessels in hyperoxic pulmonary hypertension and fibroblasts of cells in the late stage—the time of intense neomuscularization of the microvessels.