Serotonin (5HT) receptor signaling and 5HT-related agents, such as the anorexogen fenfluramine (Fen), have been associated with heart valve disease. We investigated the hypothesis that Fen may disrupt mitral valve interstitial cell (MVIC) homeostasis through its effects on mitogenesis and extracellular matrix biosynthesis. Normal and myxomatous mitral valves, both human and canine, were harvested, and primary MVIC cultures were established. 5HT caused increased phosphorylation of extracellular signal-related kinase in MVIC; Fen alone did not. However, Fen combined with 5HT increased the level of MVIC extracellular signal-related kinase, when compared with 5HT alone. In addition, MVIC mitogenesis per (3)H-thymidine ((3)HTdR) demonstrated a 5HT dose-dependent increase, with no effect of Fen alone. In contrast, Fen combined with 5HT inhibited the MVIC (3)HTdR response when compared with 5HT alone. Furthermore, fluoxetine, a 5HT transporter inhibitor, while having no effect alone, suppressed Fen-5HT (3)HTdR inhibition when administered with Fen plus 5HT. Finally, MVIC incorporations of (3)H-proline and (3)H-glucosamine, measures of extracellular matrix collagen and glycosaminoglycan respectively, were increased with 5HT alone; however, Fen did not affect MVIC glycosaminoglycan or collagen either alone or in combination with 5HT. Taken together, the ratios of (3)H-proline or (3)H-glycosaminoglycan to (3)HTdR in MVIC, normalized to 5HT alone, demonstrated a significant imbalance of extracellular matrix production versus proliferation in MVIC cultures with Fen plus 5HT exposure. This imbalance may explain in part the pathophysiology of Fen-related mitral valve disease.