Elevated levels of serotonin (5-HT) and endothelin-1 (ET-1) may be involved in cardiovascular complications of diabetes mellitus. Data suggests supra-physiological concentrations of 5-HT (10^-6 M) potentiate ET-1's ability to stimulate DNA synthesis and vascular smooth muscle cell (VSMC) proliferation in vitro via activation of mitogen activated protein kinase (p42/44 MAPK) and janus kinase 2 (JAK2) pathways. Additionally, 5-HT enhances agonist-induced contractions via p42/44 MAPK and an unknown tyrosine kinase. However, the exact mechanisms of the 5-HT/ET-1 interactions and whether these effects occur at physiological levels (10^-9 M) are unknown. Therefore, we hypothesized that interactions between 5-HT and ET-1 at physiological concentrations in VSMC enhanced activation of both p42/44 MAPK and JAK2 pathways contributing to vascular growth and contractile responses. Using rat VSMC and Western Blot analysis our data suggest no effect of acute (30 minutes) pre-incubation with 5-HT (10^-9 M) and/or ET-1 (10^-9 M) on the activation of either pathway in normal or high glucose conditions. To determine if there was altered vascular reactivity in intact vessels we tested the effects of 5-HT and ET-1 interaction using myographs to measure isometric contractions of rat thoracic aortic rings. 5-HT (10^-9 M) and ET-1 (10^-12 M) stimulate enhanced contractile responses to each other that were inhibited by JAK2 and p42/44 MAPK antagonists. Our findings demonstrate that both 5-HT and ET-1 at physiological concentrations could interact with each other and activate p42/44 MAPK and JAK2 signaling pathways to cause an increase in smooth muscle contraction which could lead to altered vascular function.