Abstract Body: Pulmonary hypertension (PH) is a disease that can lead to right ventricular (RV) fibrosis and failure due to pressure overload. The RV develops more severe fibrosis in response to pressure overload than the left ventricle (LV), which may contribute to the fact that clinical outcomes of RV failure are much worse than those found in left heart disease. As the RV has been found to contain a unique molecular and cellular makeup, we hypothesize that RV fibroblasts (RVfibs) carry out distinct mechanisms during disease that lead to exaggerated fibrosis. To characterize fibroblast activation at baseline and during PH, we utilized the Sugen-Hypoxia (SuHx) rat model (20 mg/kg SU5416 and 3 weeks of hypoxia at 10% O₂) to recapitulate PH at both the compensatory stage of cardiac remodeling (3 weeks post-hypoxia, SuHx 3(+3)) and at the point of failure (6 weeks post-hypoxia, SuHx 3(+6)), which was confirmed by echocardiography and invasive pressure catheter measurements. RVfibs and LV fibroblasts (LVfibs) were isolated by enzymatic digestion and cultured separately for analysis. We quantified fibrotic gene expression via qPCR, oxygen consumption rate (OCR) via Seahorse assay, and proliferation via CyQuant assay. We found that stimulation with 10 ng/mL TGFβ induced 1.2 times higher fold-change in periostin expression in RVfibs versus LVfibs (p<0.05 via t-test), and RVfibs increase in cell number twice as much as LVfibs over a 3 day period (p<0.05 via t-test). The proliferation rate of LVfibs isolated from SuHx 3(+3) and SuHx 3(+6) rats do not change from that of baseline LVfibs, while SuHx 3(+3) and SuHx 3(+6) RVfibs appear to match LVfibs in proliferative rate as opposed to baseline RVfibs. RNA-seq. reveals that RVfibs upregulate pathways including cell cycle checkpoints, mitosis, and RHO GTPases compared to the LV, indicating a transcriptional program favoring cell division and related signaling. RVfibs also trended towards having a greater OCR than LVfibs. Thus, RVfibs exhibit amplified proliferation, fibrotic gene expression, and OCR, suggesting an elevated capacity for fibrotic response compared to LVfibs. Future experiments will focus on determining the molecular underpinnings of this phenotype and may identify therapeutic targets for PH treatment that lessen the severity of RV fibrosis and improve clinical outcomes.