Following myocardial infarction, angiogenesis occurs as a result of thrombus formation, which permits reperfusion of damaged myocardium. Sphingosine 1-phosphate (S1P) is a naturally occurring lipid mediator released from platelets and is found in high concentrations at sites of thrombosis. S1P might therefore be involved in regulating angiogenesis following myocardial infarction and might influence reperfusion. The aims of this study were to determine the effects of S1P in human coronary arterial cell angiogenesis and delineate the subsequent mechanisms. An in vitro model of angiogenesis was developed using a co-culture of human coronary artery endothelial cells, human coronary smooth muscle cells and human fibroblasts. In this model, S1P inhibited angiogenesis and this was dependent on the presence of smooth muscle cells. The mechanism of the inhibitory effect was through S1P-induced release of a soluble mediator from smooth muscle cells. This mediator was identified as tissue inhibitor of metalloproteinase-2 (TIMP-2). Release of TIMP-2 was dependent on S1P-induced activation of Rho kinase and directly contributed to incomplete formation of endothelial cell adherens junctions. This was observed as a diffuse localisation of VE-cadherin, leading to decreased tubulogenesis. A similar inhibitory response to S1P was demonstrated in an ex vivo human arterial model of angiogenesis. In summary, S1P-induced inhibition of angiogenesis in human artery endothelial cells is mediated by TIMP-2 from vascular smooth muscle cells. This reduces the integrity of intercellular junctions between nascent endothelial cells. S1P might therefore inhibit the angiogenic response following myocardial infarction.