Serotonin (5-HT) stimulates tyrosine phosphorylation and proliferation of bovine pulmonary artery smooth muscle cells (SMC) through its active transport (Lee et al, 1991). The present studies show that 5-HT also rapidly elevates O₂^·− formation by these cells within 10 minutes as measured by a lucigenin-enhanced chemiluminescence assay. The O₂^·− free radical quencher, Tiron, and N-acetyl-cysteine, a substrate for glutathione, block both the 5-HT-induced formation of O₂^·− and cellular proliferation. Similarly, inhibition of 5-HT transport with imipramine or treatment of cells with diphenyliodonium, a NAD(P)H oxidase inhibitor, block both 5-HT-induced elevation of O₂^·− and cellular proliferation. Alpha-hydroxyfarnesylphosphonic acid, an inhibitor of p21^ras, also blocks 5-HT-induced proliferation. Endothelial cells from the same vessel show neither 5-HT-induced proliferation nor stimulation of O₂^·− formation. We conclude that 5-HT induced cellular proliferation of SMC through signaling pathways that utilize its transport system and O₂^·− formation.