Acetylcholine (ACh) is found in the nervous system, and also in other cell types (endothelium, lymphocytes, epithelial cells and blood cells) which are globally termed the nonneuronal cholinergic system. In this study we investigated the expression and subcellular localization of acetylcholinesterase (AChE), in endothelial cells (ECs). Our results show the expression of the 70kDa AChE in both cytoplasmic and nuclear compartments. We also describe, for the first time, a nuclear and cytoskeleton-bound AChE isoform with approximately 55 kDa detected in ECs. This novel isoform is decreased in response to Vascular Endothelial Growth Factor (VEGF) via the proteosome pathway and it is downregulated in human leukemic T-cells as compared to normal T-cells, suggesting that decreased expression of the 55 kDa AChE protein may contribute to an angiogenic response and associate with tumorigenesis. Importantly,we show that its nuclear expression is not endothelial-cell specific but also evidenced in non-neuronal and neuronal cells. Concerning neuronal cells, we can distinguish an exclusively nuclear expression in postnatal neurons in contrast to a cytoplasmic and nuclear expression in embryonic neurons,suggesting that the cell compartimentalization of this new AChE isoform, is changed during nervous system development. Overall, our studies suggest that the 55 kDa AChE may be involved in different biological processes such as neural development, tumor progression and angiogenesis.