Acetylcholine (ACh) is detected in a variety of non-neuronal cells where it acts as a para/autocrine signaling molecule controlling basic cell functions such as proliferation, differentation, and maintenance of cell-cell contacts. ACh-synthesizing enzymes include choline acetyltransferase and carnitine acetyltransferase (CarAT). ACh is released through vesicular exocytosis or directly from the cytoplasm via organic cation transporters (OCT). Extracellular ACh binds to nicotinic (nAChR) and muscarinic receptors (MR). Degradation of ACh is performed by acetylcholinesterase and butyrylcholinesterase (BChE). Here, we have determined whether these molecules are expressed in osteoblast-like cells, by means of reverse transcription polymerase chain reaction and immunohistochemistry, focusing on nAChR subunits alpha3 and alpha5. RNA for CarAT, OCT-1, M2R, M5R, nAChR subunits alpha3, alpha5, alpha9, alpha10, beta2, beta3, and BChE were detected in human (SAOS-2) and murine (MC3T3-E1) osteoblast-like cells. Other cholinergic components were only expressed species-specifically, e.g., M3R and nAChR subunit alpha7. Immunhistochemistry localized the nAChR subunits alpha3 and alpha5 in osteoblasts in vitro and in vivo where they were up-regulated after application of bone morphogenetic protein-2 (BMP-2) during fracture healing in a rat model. Thus, the cholinergic system of osteoblast-like cells might be regulated by BMP-2 during bone remodeling. Osteoblast-like cells express all necessary enzymes, transporters, and receptors for ACh synthesis and recycling.