The naturally occurring sphingolipid, sphingosylphosphorylcholine (SPC), is a constituent of plasma lipoproteins and is also released from activated platelets. SPC may have atheroprotective effects via an action on endothelial cells. However, on vascular smooth muscle (VSM) cells, SPC can initiate proliferation and vasoconstriction. It may therefore have a role in the development of vascular disease. We have recently shown that SPC can activate p38 mitogen-activated protein kinase (MAPK) in VSM indicating a potential role as a pro-inflammatory factor. Such a mechanism of action could contribute to a pro-atherogenic effect. The aim of the current study was to determine if SPC could induce the release of inflammatory proteins from VSM cells and delineate the underlying mechanisms. A7r5 cells (rat aortic VSM cell line) were incubated with 10 μM SPC for 24 hours at 37οC. Medium was removed and subjected to an inflammatory antibody array consisting of antibodies to 20 chemokines and cytokines. Compared to control, binding to only one antibody was significantly increased; monocyte chemoattractant protein-1 (MCP-1). Enzyme-linked immunosorbent assays (ELISA) for MCP-1 were used to confirm the results of the array. Conditioned medium from SPC-treated A7r5 cells revealed an increase in MCP-1 release (200 μg/ml) compared to control (75 μg/ml) (n=3). Lipopolysaccharide (25 μg/ml) used as a positive control, increased MCP-1 release to 415 μg/ml. Interestingly, the structurally-related sphingolipid, sphingosine 1-phosphate, did not increase MCP-1 release from A7r5 cells. The promoter regions for the MCP-1 gene contain consensus sequences for 2 transcription factors associated with inflammatory responses, nuclear factor-κB (NF-κB) and CCAAT enhancer binding protein (c/EBP). To determine whether SPC can activate either or both of these transcription factors, electromobility shift assays (EMSA) were carried out on A7r5 cells incubated with 10 μM SPC for 1 hour. SPC-treated cells showed an increased DNA-protein binding with oligonucleotides specific for a c/EBP consensus sequence. EMSA to determine DNA-binding of activated NF-κB revealed that SPC also activates NF-κB in VSM cells. For both c/EBP and NF-κB, activation by SPC was dependent on p38MAPK phosphorylation, as observed by pre-incubation with the p38MAPK inhibitor, SB203580. In conclusion, SPC can induce release of the inflammatory chemokine, MCP-1, from VSM cells. This may occur via activation of the pro-inflammatory transcription factors, NF-κB and c/EBP, leading to increased gene and protein expression of MCP-1. This SPC-induced inflammatory response may play a role in vascular disease.