Pulmonary hypertension is associated with abnormal connective tissue deposition in the media of pulmonary arteries. Lobar arteries from calves maintained for up to 15 days at simulated high altitudes showed a 35% increase in collagen and a >40% increase in crosslinked elastin per μg protein. Labeling of artery tissue with [14C]proline revealed a nearly twofold increase in relative collagen synthesis. There was increased incorporation into Types I, III, IV, and V collagen with an increase in the proportion of newly synthesized Type IV collagen. Quantitation of collagen mRNA by slot-blot assay demonstrated increased levels of Types I and IV collagen message. In addition, medial smooth muscle cells isolated from the hypertensive calves demonstrated a nearly twofold increase in relative collagen synthesis, a twofold increase in the accumulation of newly synthesized collagen per μg DNA, and increased levels of Types I and IV collagen mRNA. Exposure of pulmonary artery smooth muscle cells, adventitial cells, and fetal calf ligament fibroblasts to conditioned calf serum harvested from cultures of medial cells from hypertensive animals increased their levels of collagen as well as elastin mRNA. These studies suggest that the increased production of collagen in hypertensive arteries is mediated at a pre-transitional level by soluble factor(s) generated by medial smooth muscle cells.