Formation of new vessels from pre-existing capillaries demands extensive reprogramming of endothelial cells through transcriptional and post-transcriptional events. We show that 120 protein spots in a two-dimensional isoelectric focusing/electrophoretic analysis were affected during vascular endothelial growth factor-A-induced endothelial cell tubular morphogenesis in vitro, as a result of changes in charge or expression level of the corresponding proteins. For about 22% of the spots, the protein products could be identified, of which several previously have been implicated in cytoskeletal reorganization and angiogenesis. One such protein was heat shock protein 27, a chaperone involved in beta-actin rearrangement that was identified as regulated in degree of serine phosphorylation. We also identified regulation of chloride intracellular channel 4 (CLIC4), the expression of which decreased during tubular morphogenesis. CLIC4 was expressed at high levels in resting vessels, whereas expression was modulated during pathological angiogenesis such as in tumor vessels. The subcellular localization of CLIC4 in endothelial cells was dependent on whether cells were engaged in proliferation or tube formation. Antisense- and small interfering RNA-mediated suppression of CLIC4 expression led to arrest in tubular morphogenesis. Our data implicate CLIC4 in formation of a vessel lumen.