Muscarinic acetylcholine receptors (mAChR) are members of the G-protein coupled receptor family. These receptors play key physiological roles and changes in their expression and/or function are involved in several diseases. We had previously demonstrated that mAChR expression is up regulated in three different cell lines derived from distinct murine mammary adenocarcinomas that spontaneously arose in BALB/c female mice, in comparison with normal murine mammary cells. Stimulation of mAChR with the muscarinic agonist carbachol (CARB) potentiated different steps of tumor progression. We here evidence that similarly to previous results obtained in mice, human breast tumor homogenates over expressed mAChR in comparison with normal breast tissue. Thus, to test the muscarinic actions on human breast adenocarcinoma cells we investigate the effect of CARB on MCF-7 cells proliferation and neovascular response. Particularly we observe that: CARB stimulates tumor cells proliferation, being 10(-9) M the maximal effective dose for the muscarinic agonist. This action was due to M3 and M1 receptors activation being nitric oxide synthase (NOS) its effector enzyme via phospholipase C and protein kinase C signaling pathway. NOS1 and NOS3 isoforms are expressed in MCF-7 cells and its activation by CARB triggers nitric oxide synthesis and vascular endothelial growth factor expression increasing blood vessels formation induced by mammary tumor cells in vivo. We can conclude that nonneuronal cholinergic system activation stimulates MCF-7 tumor cells growth and neovascular response promoting tumor progression.