Motivation: Chronic hypoxia can induce pulmonary vascular smooth muscle cell (SMC) proliferation, which contributes to blood vessel constriction and lumen narrowing, leading to pulmonary hypertension. Perlecan is the major heparan sulfate proteoglycan in the basement membrane. The heparan sulfate chains of perlecan have been shown to inhibit SMC proliferation in the carotid artery. In this study, we examine if perlecan heparan sulfate can inhibit pulmonary SMC proliferation induced by hypoxia. Methods: Mice with heparan sulfate deficient perlecan (Hspg2Δ3/Δ3) were compared to wild-type (Wt) controls. 8-11 week-old female mice were subjected to hypoxia (10% O2) for 7 weeks. Controls were kept in normoxia. This generated 4 groups, Hspg2Δ3/Δ3 versus Wt, with or without hypoxic treatment. Hemoglobin levels and the right ventricle/left ventricle+septum weight ratio (RV/(LV+S)) were measured. Serial sections from lung tissue were obtained for immunohistochemical and morphological analysis. Degree of muscularization of intra-acinar vessels was determined by double staining for von Willebrand factor and smooth muscle α-actin, followed by manual counting. Proteins and total RNA were isolated from whole lung homogenate. Genotype and treatment were blinded for all data acquisition. Results: To our surprise, at baseline (normoxia) Hspg2Δ3/Δ3 mice have a significantly lower proportion of muscularized intra-acinar vessels compared to Wt. After 7 weeks of hypoxia, a significant increase in muscularized vessels was found in both Hspg2Δ3/Δ3 (2.8 fold) and Wt mice (1.5 fold). The RV/(LV+S) ratio, as a measurement of right ventricular hypertrophy, is the same in Hspg2Δ3/Δ3 and Wt at normoxia, and it increases significantly following hypoxia, with no difference between the genotypes. For Wt mice, RNA expression of FGF-2 and PDGF-B are both upregulated in hypoxia. In Hspg2Δ3/Δ3 mice, FGF-2 is upregulated already in normoxia, possibly as a compensation for increased elimination. For PDGF-B the increase following hypoxia is higher in Hspg2Δ3/Δ3 mice compared to Wt. Conclusions: The lower proportion of muscularized vessels at baseline in Hspg2Δ3/Δ3 mice is interesting and needs to be further investigated. With hypoxia treatment, the relative difference in the increase of muscularization, 2.8-fold in Hspg2Δ3/Δ3 versus 1.5-fold in Wt, indicates that Hspg2Δ3/Δ3 mice are more reactive in their vessels and therefore may be more vulnerable to hypoxia. The differences in muscularization may be caused by defective sequestration of heparin binding growth factors in the extracellular matrix and thereby also different regulation of those growth factors in response to hypoxia. The results suggest that perlecan heparan sulfate chains are important for the regulation of muscularization of pulmonary vessels during vessel development and during hypoxia.