A proteomic approach was applied to explore the signalling pathways elicited by lowering O(2) in endothelial cells. Endothelial cells isolated from native umbilical cords were subjected to 21, 5, or 1% O(2) for 24 h. 2-D PAGE was performed and candidate proteins were identified using LC-MS/MS. Lowering of O(2) from 21 to 5% induced upregulation of cofilin-1, cyclophilin A, tubulin and tubulin fragments, a fragment of glucose-regulated protein 78 (Grp78) and calmodulin. The upregulation of Grp78 suggested that ER stress proteins were altered and indeed Grp94 and caspase 12 expression were increased in cells exposed to 5% O(2). The presence of ER stress is also supported by findings of blunted caffeine-evoked ER calcium release in cells exposed to 5 and 1% O(2). Exposure to 1% O(2) caused increases in cofilin-1, cyclophilin A, and caspase 12 as well as a decrease of beta-actin, but it did not alter the expression of calmodulin, tubulin, Grp78, and Grp94. Incubation with CoCl(2), a stabilizer of the hypoxia-inducible factor, increased the expression of several of the proteins. The present investigations reveal that lowering O(2), probably in part through hypoxia-inducible factor, alter the expression of a series of proteins mainly involved in cytoskeletal changes (e.g. cofilin-1, tubulin, and beta-actin) and in ER stress/apoptosis (e.g. Grp78/94, caspase 12, and cyclophilin A).