A role may be attributed to cholinergic system in the development of hematological diseases. Recent studies demonstrated a significant increase in AChE activity together with muscarinic receptor expression in normal human peripheral blood lymphocytes as an early response to various stimuli (Battisti et al 2009). K562 cell line was established from a patient with chronic myeloid leukemia (CML) in terminal blast crisis. We have previously demonstrated the presence of muscarinic receptor subtypes namely M2, M3 and M4 in K562 line (Cabadak et al., 2010). We hypothesized that cholinergic agonist ACh is involved in cell proliferation in CML through muscarinic receptors. In this present study, we aimed to demonstrate the mechanisms of ACh and carbachol (CCh) in cell proliferation. In order to track the roles of ACh we used hemicholinium, a choline uptake inhibitor, eserine hemisulfate, an acetylcholine esterase inhibitor and vesamicol a vesicular choline uptake inhibitor. We also made experiments with atropine the non-specific muscarinic antagonist to show the contribution of muscarinic receptors. K562 cell counts were performed in response to CCh stimulation where proliferation and cell viability were evaluated by the trypan blue exclusion test and BrDU labeling. AChE activity was determined by QuantiChrom TM Acethylcholine Assay kit. Choline acetyl transferase (ChAT) and AChE levels were quantified by using Western Blotting. In our experiments we detected an inhibition of K562 cell count after 48 hr incubation in response to CCh at a concentration of 1 uM. Non-specific receptor antagonist atropine failed to reverse the inhibitory roles of CCh and ACh. Eserine also acted similarly on K562 cells and inhibited cell proliferation and atropine could not affect this inhibition (Table 1). Neither hemicholium nor vesamicol affected the cell counts. The effects observed with eserine is specifically dependent on ACh since we demonstrated a dose-dependent decrease in AChE activity concurrently. The experiments performed with vesamicole and hemicholinium imply that ACh secreted form other cells decrease proliferation of K562 cells. In conclusion, all these results may imply that extrinsic ACh activity affects cell proliferation in CML through nicotinic mechanisms, but ACh syhthesized within the cell elicits muscarinic receptor mediated effects.