Serotonin (5-HT) and fibronectin (FN) have been associated with pulmonary hypertension (PH). We previously reported that FN is posttranslationally modified by tissue transglutaminase (TGase) to form serotonylated FN (s-FN) in pulmonary artery smooth muscle cells and that serotonylation stimulates their proliferation and migration, hallmarks of PH. We hypothesized that s-FN and its binding to TGase are elevated in human and experimental PH. To assess this hypothesis, FN isolation and electrophoretic, immunoblotting, and densitometric techniques were used. Mean ratio of serum s-FN to total FN level (s-FN/FN) was elevated in 19 consecutive pulmonary arterial hypertension (PAH) patients compared with 25 controls (0.3 ± 0.18 vs. 0.05 ± 0.07, P < 0.001). s-FN/FN also was increased in lungs of mice and rats with hypoxia-induced PH and in rats with monocrotaline-induced PH. In mice, the increase was detected at 1 wk of hypoxia, preceding the development of PH. Hypoxic rats had elevated serum s-FN/FN. Enhanced binding of TGase to its substrate FN occurred in serum from patients with PAH (mean 0.50 ± 0.51 vs. 0.063 ± 0.11, P = 0.002) and s-FN/FN and TGase-bound FN were highly correlated (R(2) = 0.77). TGase-bound FN also was increased in experimental PH. We conclude that increased serotonylation of FN occurs in human and experimental PH and may provide a biomarker for the disease.